Therapeutic effects of aqueous extract of bioactive active component of Ageratum conyzoides on the ovarian-uterine and hypophysis-gonadal axis in rat with polycystic ovary syndrome: Histomorphometric evaluation and biochemical assessment.

Background: Polycystic ovary syndrome (PCOS) is an endocrine disorder, affecting women of reproductive age. Ageratum conyzoïdes (AGC) is used traditionally in the treatment of fever, rheumatism, and ulcer. This study investigates the effects of AGC on ovarian-uterine in PCOS rats.
Methods: Female rats were randomized into four groups (n = 6). Group A control received 2 ml distilled water. Group B received a single dose of 4 mg/kg body weight (bwt) i.p estradiol valerate (EV). Group C received 500 mg/kg bwt AGC and group D received a single dose of 4 mg/kg bwt i.p EV followed by 500 mg/kg bwt AGC orally for 30 days. Parameters tested include follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), estradiol (E2), progesterone (P), C-reactive protein (CRP), interleukin (IL)-6, IL-18 and tumor necrosis factor (TNF)- α, malondialdehyde (MDA), superoxide dismutase (SOD), Catalase (CAT), total protein (TP), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), and ovary and uterus histomorphometric.
Results: Ageratum conyzoides decrease insulin resistance, obesity indices, TC, TG, LDL, MDA, T, LH, FSH, CRP, IL-6, IL-18, and TNF- α in PCOS rats. And increase HDL, E2, P, TP, CAT, and SOD in PCOS rats. AGC improved ovary and uterus histo-architecture, tertiary, and Graafian follicles, corpus luteum and endometrial thickness increased,and cystic and atretic follicles decreased.
Conclusion: Ageratum conyzoides improved insulin sensitivity, antioxidant activities, hormonal imbalance, inflammatory makers, and histological changes in PCOS rats. Therefore AGC can be used as a potential adjuvant agent in the treatment of PCOS.

Analysis of variance

Enzyme-linked Immunosorbent Assay

Estradiol Valerate

Fasting plasma glucose

Fasting insulin

Follicle-stimulating hormone

Glutathione peroxidase enzyme

Hematoxylin and eosin

Homeostasis model assessment of insulin resistance

Low-density lipoprotein cholesterol

Luteinizing hormone

Malondialdehyde

Polycystic ovarian syndrome

Standard error of the mean

Superoxide dismutase enzyme

Introduction

Polycystic ovarian syndrome (PCOS) is a gynecological endocrine disorder that affects women of reproductive age [1,2]. Hyperandrogenism is characterized by a hormonal imbalance, chronic menstrual dysfunction (oligo- or anovulation), impaired fertility, hirsutism (excessive body hair growth), acne, obesity, diabetes mellitus, metabolic disturbances (dyslipidemia, hyperinsulinemia, insulin resistance, and type 2 diabetes), increased incidence of endometrial hyperplasia and cancer, and policy [3]. Polycystic ovary syndrome affects around 6–20% of women of reproductive age, and its incidence is on the rise and is the leading cause of female infertility [4,5]. Polycystic ovary syndrome can disrupt ovulation and can lead to infertility; as a result, it is not surprising that infertility affects 70–80% of women with PCOS [6]. In addition, PCOS has been linked to an increased risk of miscarriage, anxiety, and sadness [7]. Also, PCOS is commonly regarded as one of the most significant risk factors for type I endometrial carcinoma (EC) [8]. The primary cause of this elevated risk is thought to be prolonged estrogen exposure to the endometrium as a result of anovulation [9]. As a result, PCOS-related hormonal imbalances can disrupt endometrial tissue homeostasis and stimulate cell growth [10]. Continuous estrogen exposure to the endometrium in humans can cause endometrial hyperplasia [11]. Progesterone protects the uterus against estrogen-induced uterine development and proliferation [12]. The primary cause of glucose metabolic disease is insulin resistance (IR), which is also thought to be a key factor in the development of PCOS, which is strongly linked to concurrent metabolic difficulties [3].Low insulin sensitivity, often known as IR, is a syndrome that causes compensatory hyperinsulinemia and plays a significant role in the etiology of type 2 diabetes [4]. Increased levels of testosterone and luteinizing hormone are released as a result of hyperinsulinemia, which disrupts the uptake and utilization of glucose and results in prolonged anovulation [9].Allopathic medications such as clomiphene citrate, metformin, letrozole, tamoxifen, and troglitazone are now the most well-known treatments for PCOS [13]. Hot flushes, arthritis, joint or muscular discomfort, and psychological side effects such as irritability, mood swings, sadness, and bloating are all common adverse effects of allopathic drugs [14]. Since obesity affects the clinical presentation of PCOS, environmental variables including eating habits are crucial in both the prevention and treatment of PCOS. Saturated and trans fatty acid intake will be reduced, and any deficits in vitamins D, chromium, and omega-3 should also be taken into consideration [9]. According to reports, PCOS can result from a lipid-induced proinflammatory state; therefore, a healthy diet with an appropriate macronutrient distribution looks like a suitable choice. The anti-inflammatory properties of the Mediterranean diet (MD) and its link to weight loss make it a suitable dietary treatment for PCOS. Regular consumption of unsaturated fats, low-glycemic carbs, fiber, vitamins, antioxidants, and a modest intake of animal protein is the basis of the MD [9].Alternative therapies such as Laparoscopic ovarian surgery, acupuncture, naturopathy, and herbal medications are gaining popularity as a result of the negative side effects induced by allopathic drugs [15]. The use of plants as traditional medicine has shifted the paradigm away from synthetic drugs and toward natural products (returning to nature) [16,17]. Herbal medical resources are regarded as an important aspect of the natural world [18,19]. Novel active ingredients, particularly those of natural origin, have piqued researchers' curiosity for years due to their unique chemical structures and powerful bioactivities [20,21]. The fact that the majority of therapeutic agents licensed in the previous century were derived from plants or natural sources demonstrates the relevance of plant actives in the creation of innovative medications [22,23]. Ageratum conyzoides (AGC) is a medicinal plant that is useful against various ailments such as fever, rheumatism, head arch, colic, burn wounds, dyspepsia, eye problem, uterine disorder, pneumonia, ulcers e.t.c. and may include physiologically active substances that are beneficial to one's well-being [24]. It is an upright, annual herbaceous herb that is native to tropical America, particularly Brazil, and across Africa, save for arid environments [25]. Different tribes in Nigeria have different names for it. The Igedes from the central belt, Yorubas from the southwest, and Igbos from the southeast of the country, for example, refer to it as “ufuopioko,” “imiesu,” and “nriewu,” respectively [26]. The plant grows close to human settlements and thrives in soils rich in nutrients, minerals, and moisture [27]. It can be found in and near waste spaces, gardens, meadows, disturbed habitats, forest borders, watercourses, ruined sites, and other locations, ranging in elevation from sea level to mountainous terrain [28]. Due to its quick growth rates, short life cycles, drought tolerance, allelopathy, increased competitive powers, and higher reproductive capacity, AGC successfully invades natural environments [29]. Ageratum conyzoides is a fragrant plant with a long history of use in traditional medicine throughout the world [30]. Several secondary metabolites from AGC have been identified and described, including flavonoids, alkaloids, chromene, terpenoids, coumarins, and sterols, among others [31,32]. Radioactive, antidiabetic, antibacterial, anti-inflammatory, antioxidant, anticancer, and wound healing effects are all claimed for these secondary metabolites [33]. Based on reports of the various beneficial medicinal potential of the bioactive components of AGC, the current study aims to investigate the possible therapeutic and ameliorative effects of aqueous extract of Ageratum conyzoides on the ovarian-uterine and hypophysis gonadal axis in a rat with polycystic ovary syndrome.

Materials and methods

Chemical and reagent

Estradiol valerate(NAMAN PHARMA DRUGS, Mumbai.,Mfg.Lic.No.: L/19/2186/MB) was purchased from FEGTOCHI Pharmacy, Akure, Nigeria. Pentobarbital sodium (40 mg/kg, i.p.) was purchased from PASCAL (Nigeria). Thiobarbituric acid and reduced glutathione were purchased from Sigma-Aldrich Corp. (St. Louis, MO USA). Assay kits for protein are products of Randox Laboratories Limited (Co Antrim, United Kingdom) while those of testosterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol, progesterone, and leptin were obtained from Monobind Inc. (California, USA). All other reagents used were of analytical grades.

Plant material and extraction of the plant material

Ageratum conyzoides were harvested in Ondo in October 2021, (Southwest region, Nigeria). A botanist, Mr. Omomoh Bernard, validated the plant at the Herbarium division of the Federal University of Technology Akure, Ondo State Nigeria, where a voucher specimen (FUT/2021/012)was filed for reference purposes. Fresh and clean Ageratum conyzoides leaves were air-dried (in the shade), and ground. One kilogram (1 Kg) of the powder was macerated for 12 h in 10 liters of distilled water before being filtered using Whatman paper number 4 [34]. This initial filtrate was kept in the fridge until it was needed. The same amount of distilled water (10 L) was poured into the residue for an additional maceration of 12 h. Following the filtration of this second macerate, the filtrate obtained was added to the first filtrate. The whole was freeze-dried and a total dry mass of 53.53 g of the aqueous extract was obtained. This extract was kept at 4 °C in an airtight container until use [35].

Phytochemical screening

The aqueous leaf extract of Ageratum conyzoides was analyzed qualitatively and quantitatively using a variant of Soni and Sosa's approach [36]. In modifying the study by Grindberg and Williams [37] high-performance liquid chromatography was used to measure the vitamins. The content of minerals such as sodium, calcium, potassium, iron, zinc, and phosphorus was determined using a modified version of the Akubugwo et al. technique [38].

Experimental animals

In this study, twenty male Sprague Dawley rats (weighing 160–200 g) were employed as experimental animals. At the Animal House of the Department of Human Anatomy, Federal University of Technology, Akure, Nigeria, the rats were kept in plastic cages and kept on a 12:12-h light/dark schedule at room temperature. Rat pellets (Vital Feeds Nigeria Limited, Jos, Nigeria) and unlimited water was given to the rats. The rats were acclimatized for two weeks before treatment and handled humanely following the NIH Guidelines for the Care and Use of Laboratory Animals authorized animal experimental protocols [39].

Vaginal smears

Each animal's reproductive cycle was determined by collecting vaginal smears daily (8–10 a.m.). Proestrus is characterized by the preponderance of nucleated epithelial cells (the first stage). Cornifield squamous epithelial cells, which appear in clusters, were present during estrus (the second stage). Metestrus (the third stage) is made up of a variety of cell types, with leukocytes predominating with a few nucleated and/or cornifield squamous epithelial cells thrown in for good measure. Diestrus (the fourth stage) is primarily made up of leukocytes [40,41].

Experimental design

Twenty-four (24) sexually mature female rats were randomized into four groups (A,B,C, and D) of six (n = 6) rats each. Group A served as control and received 2 ml of distilled water. Group B received a single dose (4 mg/kg body weight i.p injection of estradiol valerate (EV) on the first day, Animals in groups C received 500 mg/kg body weight of Ageratum conyzoides extract orally, and group D was treated with a single dose (I.P 4 mg/kg body weight) injection of EV on the first day followed by 500 mg/kg body weight of Ageratum conyzoides extract orally. All administrations were done once daily for 30 days.

Assessment of the rat's anthropometric parameters

During the experiment, the rats' initial and end body weight and length were measured daily at roughly 16:00 clocks. To assess obesity in animals, the body mass index (BMI) and the Lee index were determined. By dividing the weight (g) by the length (cm2), the BMI was computed [43]. The distance between the snout and the anus of rats was used to determine body length. Meanwhile, the Lee index for each animal was calculated by multiplying the complete expression by 1000 and dividing the cube root of the body weight (g) by the naso-anal length (Cm) i.e [LI = body weight (g)1/3 x1000/body length (cm)]. If the Lee index score was greater than 310, rats were termed fat [44,45].

Determination of insulin resistance

The Ultra-Sensitive Rat Insulin ELISA kit (Cloud-Clone Corp., Houston, USA) was used to measure serum insulin concentrations, as reported by Nurdiana et al. [46]. According to Shen et al. [47], the homeostatic model assessment of insulin resistance (HOMA-IR) was calculated as follows:

Sample collection

Animals were permitted to fast for 12 h at the end of the trial. The Accu-check Advantage II Blood Glucose Monitor (Roche Mannheim, Germany) was used to measure fasting blood glucose. The highest limit of detection was 33.3 m. After 24 h, animals were sedated with pentobarbital sodium (40 mg/kg, i.p.) [48]. To separate serum, blood samples were taken from the jugular vein and centrifuged for 10 min at 3000 rpm. After then, the samples were kept at −20 °C until they were analyzed. The cervical dislocation was used to slaughter the animals and the ovary and uterine tissues were subsequently separated. After that, the tissues were rinsed with regular saline. One portion of the tissues was preserved in 10% phosphate-buffered formalin for histological investigation [49]. The remaining portion was homogenized in 0.1 M ice-cold Tris-HCl buffer (1/10 w/v, pH 7.4) for biochemical analysis.

Ovarian homogenate preparation

Using a Potter Elvehjem homogenizer, each rat's left ovary was homogenized individually in 50 mm Tris–HCl buffer (pH 7.4) containing 1.15 percent KCl to generate a 20 percent (1/5 w/v) tissue homogenate. The homogenates were then centrifuged for 10 min at 10,000 g in a chilled centrifuge (4 °C). The supernatants were collected and used to determine total oxidant and antioxidant activity [50].

Determination of lipid profile

The concentration of the respective parameters; Fasting glucose, triglycerides (TG), total cholesterol (TC), and high-density lipoprotein (HDL) was read directly using a chemistry analyzer (spectrophotometer), whereas the concentration of VLDL was extrapolated by dividing the respective concentration of TG by 5 while LDL-cholesterol estimated using the method by Friedewald et al. [51] that LDL-C = TC -(HDL-C) – Triglycerides/5.

Determination of serum hormone and inflammatory markers

Enzyme-linked immunosorbent assay (ELISA) kits were used according to the manufacturer's instructions to measure the serum concentrations of hormones [Follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), estradiol (E2), progesterone (P), inflammatory factors [C-reactive protein (CRP), interleukin (IL)-6, IL-18 and tumor necrosis factor (TNF)- α].

Determination of ovarian malondialdehyde (MDA)

Ovarian levels of malondialdehyde (MDA) were determined by the method of Wilbur et al. [52]and Adelakun et al. [53]which is based on the reaction with thiobarbituric acid (TBA) at temperature of 90–100 °C. In the TBA test reaction, MDA or MDA-like substances and TBA react with the production of a pink pigment having an absorption maximum at 532 nm. Tissue level of MDA was determined using the following formula: [MDA] = DO/ε.l.m, where [MDA] = concentration of MDA (nM/mg of tissue); DO = absorbance of the sample - absorbance of the reagent blank; ε = molar extinction coefficient (1.56.10–4 nM− 1 cm− 1); l = path length (1 cm); m = mass of the tissue collected for homogenization (mg).

Determination of ovarian superoxide dismutase (SOD)

Ovarian superoxide dismutase was assayed by the method of Asada et al. [54], which involves the inhibition of photochemical reduction of nitro blue tetrazolium (NBT) at pH 8.0. A single unit of enzyme is defined as the quantity of superoxide dismutase required to produce 50% inhibition of photochemical reduction of NBT. The absorbance was read at 580 nm against a blank using a UV–Vis spectrophotometer. The activity was expressed as U/mg protein.

Determination of ovarian catalase activity

Catalase activity was estimated by the method of Sinha [55] which is based on the decomposition of H2O2 into water. The concentration of undecomposed H2O2was evaluated using a calibration curve established from a standard solution (50 mM H2O2). Tissue catalase activity was determined as follows: C. A = DO/a.t.p, where: C. A = catalase activity (mole of H2O2/min/g of total proteins); DO = absorbance of the sample - absorbance of the reagent blank; a = slope of the calibration curve; t = reaction time (1 min); p = ovarian total protein level (g).

Determination of ovarian total proteins (TP)

Ovarian levels of total peroxidases were determined by the method of Habbu et al. [56] and the method description partly reproduces their wording. Briefly, ovarian homogenate (0.5 ml) was taken, and to this were added 1 ml KI solution (10 mM) and 1 ml sodium acetate (40 mM). The absorbance of potassium iodide was read at 353 nm, which indicates the amount of peroxidase. Then 20 μl of H2O2 (15 mM) was added, and the change in the absorbance in 5 min was recorded. Units of peroxidase activity were expressed as the amount of enzyme required to change the optical density by 1 unit per min. The specific activity expressed in terms of units per g of proteins was deduced by the law of Beer-Lambert [57,58] as follows: C = DO/ε.l.p, where C = concentration of ovarian total peroxidases (mM/g of total proteins); DO = optical density; ε = molar extinction coefficient (11.3 M− 1 cm− 1); l = path length (1 cm); p = ovarian total protein level (g).

Ovarian and uterine histological procedure

The ovarian and uterine tissues, fixed with 10% formaldehyde were used for histological studies. The tissues were washed overnight in running water to remove the remaining fixative. Dehydration was carried out to remove water using a series of gradually increasing concentrations of alcohol. These tissues were then cleared in xylene embedded in wax and cut in sections of 5-μm thickness using a Rotary Microtome e (micron HM 315 microtome, Walldorf, Germany). The sections were recovered from wax blocks and stained with hematoxylin and eosin (H&E) [59,60]. The sections were viewed and photographed using an Olympus light microscope (Olympus BX51, Tokyo, Japan) with an attached camera (Olympus E−330, Olympus Optical Co. Ltd., Tokyo, Japan).

Histomorphologicalassessment

Histological analyses of the ovary and the uterus were assessed from 5 μm sections of paraffin-embedded tissues following hematoxylin-eosin staining [[61], [62], [63]]. Histomorphological changes were assessed on microphotographs using a Scientico STM-50 microscope equipped with a Celestron MA411101 camera connected to a computer where the image was transferred and analyzed with the Image J1.3 software. Follicles were recognized and counted in different stages including primary (possess mitotic, cuboidal granulosa cells), preantral (presence of theca and multiple layers of granulosa cells), antral (formed an antrum), Graafian (characterized by a large follicular antrum), cystic (full of liquid) follicles, and corpus luteum. All microscopic measurements for ovary morphometry were done using an eyepiece reticle [64].

Statistical analysis

Data were analyzed using the GraphPad Prism 8.03 software and are presented as mean ± standard error of the mean (S.E.M.). Statistical significance and the difference among groups were evaluated by one-way analysis of the variance (ANOVA) followed by Tukeys’ Post Hoc test for multiple comparisons. Differences were considered significant at p < 0.05.

Plant material: Ageratum conyzoides Photo: Vijaya Shougrakpam

Results

Estrous cycle

Fig. 3 depicts the estrous cycle of control animals, which lasted an average of 5 days and included the following estrous phases: proestrus, estrus, metestrus, and two phases of diestrus. Throughout the research period, rats in the control and AGC alone groups exhibited a consistent estrus cycle of 4–5 days. However, the estrous cycle in PCOS animals was disturbed and halted, and all of the rats' estrus cycles paused following EV injection, generally during the diestrus or metestrus stages. There was cyclic appearance of the various phases of the estrous cycle in animals receiving AGC, beginning with the proestrus phase. The PCOS + AGC therapy group demonstrated better estrous cyclicity. Compared to PCOS rats, there was a higher frequency of the estrus phase and a shorter diestrus phase (see Fig. 2).

Fig. 3

Effects of Ageratum conyzoides extract on estrous cycle in PCOS rats. PCOS: Polycystic ovary syndrome, AGC; Ageratum conyzoides. Data show the most represented phase of the estrous cycle in each group, n-6. P = proestrus stage-nucleated epithelial cells, E = estrous stage-cornified cells, M = metestrus-nucleated, cornified and leucocytes, and D = diestrous stage-leucocytes.

Fig. 2

Vaginal smear figures and cell kinds. Preoestrus contains mostly epithelial cells; estrus contains mostly keratinocytes; metoestrus contains epithelial cells, keratinocytes, and leukocytes; diestrus contains mostly leukocytes (Magnification: ×100) [42].

Alkaloids, phlorotannins, flavonoids, tannins, terpenoids, cardiac glycoside, saponins, steroids, sodium, potassium, calcium, and phosphorous were found in Ageratum conyzoides aqueous crude leaves extract [Table 1]. Total saponins, total phenol, and total flavonoids had greater levels after quantitative examination than total tannins and total alkaloids. Vitamins A, C, D, and E were also in great supply [Table 2].

Table 1

Qualitative phytochemical analysis of aqueous crude extract of Ageratum conyzoides.

S/N	Phytochemicals	status
1	Cardiac glycosides	+
2	Tannins	+
3	Saponins	+
4	Steroids	+
5	Alkaloids	+
6	Flavonoids	+
7	Phenolic acid	+
8	Terpenoids	+
9	Phlobatannins	+
10	Quinones	–
11	Coumarins	–
12	Anthracene	–
13	Sodium	+
14	Potassium	+
15	Calcium	+
16	Phosphorous	+
17	Zinc	–
18	Iron	–

+ Present, – not present.

Table 2

Quantitative phytochemical analysis of aqueous crude extract of Ageratum conyzoides.

S/N	Phytochemicals	Quantity
1	Vitamin A (mg/g)	1.84
2	Vitamin C (mg/g)	1.04
3	Vitamin D (mg/g)	5.54
4	Vitamin E (mg/g)	5.11
6	Total Tannins (%)	6.11
7	Total Saponins (%)	14.32
8	Total Flavonoids (%)	9.75
9	Total Phenols (%)	16.03
10	Total Alkaloids (%)	6.34

Anthropometry parameters of the rats

In all of the experimental groups, there was a marked increase in final body weight (p < 0.05) when compared to beginning body weight. When compared to the control group, the PCOS group's final body and abdominal fat weight increased considerably (p0.05). However, as compared to the PCOS group, the AGC and PCOS + AGC groups showed a substantial reduction in body and abdominal fat weight (p < 0.05)[Table 3].

Table 3

Effect of Ageratum conyzoides extract on body, ovary, and uterine weight in Polycystic ovarian syndrome rats.

Parameters	Treatment groups
	Control	PCOS	AGC	PCOS + AGC
Initial body weight (g)	186.80 ± 10.64	188.60 ± 9.78	188.40 ± 7.38	193.20 ± 12.22
Final body weight (g)	208.40 ± 9.41¥	245.20 ± 11.00¥*	214.60 ± 9.91¥α	212.80 ± 10.94¥ α
Weight difference (g)	21.60 ± 1.81	56.60 ± 5.84*	26.20 ± 6.40α	27.60 ± 4.89α
Initial BMI (g/cm2)	0.42 ± 0.02	0.54 ± 0.03*	0.61 ± 0.04	0.59 ± 0.02
Final BMI (g/cm2)	0.46 ± 0.01	0.62 ± 0.02*	0.51 ± 0.02	0.50 ± 0.03
Initial Lee's Index	296.42 ± 4.20	342.60 ± 6.02*	292.82 ± 5.20	310.12 ± 4.22
Final Lee's Index	291.06 ± 4.01	338.22 ± 4.01*	286.32 ± 4.62	298.43 ± 4.46
Abdominal fat weight (g)	2.68 ± 0.18	3.82 ± 0.15*	2.46 ± 0.15α	2.66 ± 0.17 α
Abdominal fat weight (g/100)	1.29 ± 0.11	1.56 ± 0.03	1.16 ± 0.11α	1.26 ± 0.11
Ovary weight (g)	0.26 ± 0.02	0.12 ± 0.01*	0.26 ± 0.01α	0.22 ± 0.03α
Ovary weight (g/100)	0.12 ± 0.01	0.05 ± 0.01*	0.13 ± 0.01α	0.10 ± 0.01α
Uterus weight (g)	0.42 ± 0.03	0.22 ± 0.02*	0.43 ± 0.02α	0.41 ± 0.03α
Uterus weight (g/100)	0.20 ± 0.01	0.09 ± 0.01*	0.20 ± 0.01α	0.19 ± 0.02α

Data presented as mean ± S.E.M, n = 6 in each group, *: represent a significant difference from control, α: represent a significant difference from PCOS at p < 0.05, One-Way ANOVA. Follow by Tukey's multiple comparisons test, AGC: Ageratum conyzoides PCOS: Polycystic ovary syndrome, BMI: Body mass index.

Furthermore, in PCOS-induced rats, there was a significant rise in BMI and Lee index values as compared to controls (p < 0.05). During the trial, the mean value of the Lee index was maintained in the PCOS rats. The mean value of the Lee Index in PCOS rats was substantially reduced after treatment with AGC. Meanwhile, in groups treated with AGC and PCOS + AGC, the mean value of BMI and Lee Index decreased significantly (p < 0.05) [Table 3].

When compared to the control rats, the PCOS rats had a substantial decrease in ovarian, uterine, and uterine epithelial height (p < 0.05). In comparison to PCOS rats, AGC therapy substantially increased ovarian, uterine, and uterine epithelial height (p < 0.05). Furthermore, the AGC-treated group only had a significant increase in ovarian, uterine, and uterine epithelial height when compared to the PCOS group (p < 0.05), while the AGC-treated group had an insignificant higher value in ovarian, uterine, and uterine epithelial height when compared to the PCOS + AGC treatment group (p > 0.05) [Table 3].

Hormone profile

PCOS causes a substantial increase in testosterone, LH, and FSH levels when compared to the control group (p < 0.05). However, as compared to PCOS, post-treatment with AGC lowered the testosterone, LH, and FSH levels (p < 0.05). Furthermore, AGC treatment significantly reduced testosterone, LH, and FSH levels when compared to PCOS (p < 0.05), and there was no significant difference in testosterone, LH, or FSH between AGC and PCOS + AGC (p > 0.05). There was no significant difference in LH/FSH ratio between the control and the experimental groups (p > 0.05) [Table 4].

Table 4

Effect of Ageratum conyzoides extract on the hormonal assay of polycystic ovarian syndrome rat.

Parameters	Treatment groups
	Control	PCOS	AGC	PCOS + AGC
Testosterone (ng/dL)	2.01 ± 0.29	11.86 ± 1.76*	2.29 ± 0.16α	3.44 ± 0.48α
Luteinizing hormone (IU/L)	5.73 ± 1.53	17.47 ± 3.34*	6.99 ± 1.47α	7.45 ± 1.49α
Follicle-stimulating hormone (IU/L)	25.09 ± 5.29	79.07 ± 12.79*	22.31 ± 4.96α	29.59 ± 6.35α
Progesterone (ng/mL)	25.07 ± 4.17	5.48 ± 1.72*	22.66 ± 4.37α	21.20 ± 3.92α
Estradiol (pg/mL)	13.18 ± 1.65	2.53 ± 0.43*	12.71 ± 1.54 α	11.71 ± 1.51 α
LH/FSH Radio	0.24 ± 0.06	0.23 ± 0.03	0.36 ± 0.07	0.28 ± 0.05

Data presented as mean ± S.E.M, n = 6 in each group, *: represent a significant difference from control, α: represents significant difference from PCOS at p < 0.05, One-Way ANOVA. Follow by Tukey's multiple comparisons test, AGC: Ageratum conyzoides PCOS: Polycystic ovary syndrome.

In PCOS, progesterone and estradiol levels were significantly lower than in the control group (p < 0.05). In contrast, when compared to PCOS rats, the group treated with AGC and PCOS + AGC exhibited substantial increases in progesterone and estradiol (p < 0.05). Between control, AGC, and PCOS + AGC (p > 0.05), there was no significant difference in progesterone and estradiol [Table 4].

Lipid profile

The cholesterol and triglycerides

In PCOS rats, cholesterol and triglyceride levels were significantly greater (p < 0.05) than in the control group. However, as compared to PCOS-induced rats, post-treatment with AGC substantially reduced cholesterol and triglyceride levels (P < 0.05). Treatment with AGC reduces cholesterol and triglyceride levels significantly when compared to the PCOS group ( 0.05) [Table 5] (see Table 6).

Table 5

Effect of Ageratum conyzoides extract on lipid profile in polycystic ovarian syndrome rat.

Parameters	Treatment groups
	Control	PCOS	AGC	PCOS + AGC
Total cholesterol (mg/dl)	50.23 ± 8.77	85.66 ± 4.79*	45.54 ± 8.46α	54.82 ± 8.17α
Triglycerides (mg/dl)	50.35 ± 9.64	75.27 ± 10.0*	46.14 ± 9.22α	54.13 ± 9.88α
HDL-C (mg/dl)	33.15 ± 5.80	17.05 ± 3.14*	36.57 ± 5.97α	31.12 ± 5.54α
LDL-C (mg/dl)	18.86 ± 3.31	38.71 ± 5.62*	16.90 ± 3.06α	21.18 ± 3.68α
VLDL-C (mg/dl)	10.07 ± 1.93	15.06 ± 2.00*	9.230 ± 1.84α	10.83 ± 1.98α
LDL-C/HDL-C	0.58 ± 0.06	1.914 ± 0.39*	0.46 ± 0.05α	0.70 ± 0.07α
TC/HDL-C	1.73 ± 0.38	5.22 ± 2.15*	1.37 ± 0.29α	2.01 ± 0.44α
TG/HDL-C	2.44 ± 1.11	5.55 ± 2.68*	1.53 ± 0.49α	2.17 ± 0.70α

Data presented as mean ± S.E.M, n = 6 in each group, *: represent a significant difference from control, α: represents significant difference from PCOS at p < 0.05, One-Way ANOVA. Follow by Tukey's multiple comparisons test, AGC: Ageratum conyzoides PCOS: Polycystic ovary syndrome.

Table 6

Effect of Ageratum conyzoides extract on the biochemical assay of polycystic ovarian syndrome rat.

Parameters	Treatment groups
	Control	PCOS	AGC	PCOS + AGC
Malondialdehyde (nmol/ng protein)	2.03 ± 0.30	9.01 ± 1.26*	1.91 ± 0.28α	2.49 ± 0.46α
Superoxide dismutase (μmole/g total proteins)	12.18 ± 2.21	3.79 ± 0.86*	9.92 ± 2.02α	14.41 ± 2.09α
CAT (U/mg protein)	25.91 ± 5.87	6.08 ± 1.58*	23.08 ± 5.52α	23.77 ± 5.62α
Total peroxidases (mM/g of total proteins)	31.12 ± 3.08	11.07 ± 2.35*	28.63 ± 3.23α	27.90 ± 2.38α

Data presented as mean ± S.E.M, n = 6 in each group, *: represent a significant difference from control, α: represents significant different from PCOS at p < 0.05, One-Way ANOVA. Follow by Tukey's multiple comparisons test, AGC: Ageratum conyzoides PCOS: Polycystic ovary syndrome.

HDL-C, LDL-C, and VLDL

There was observed a significant increase (P < 0.05) in LDL-C and VLDL-C in the PCOS group as compared to the control group. But, the level of HDL-C levels reduce significantly in the PCOS group compared to the control (p < 0.05). Treatment with AGC after induction of PCOS increased HDL-C and decreased VLDL-C and HDL-C when compared to that of PCOS-induced rats [Table 5].

LDL-C/HDL-C, TC/HDL-C and TG/HDL-C

Also, a significant increase (P < 0.05) in ratios of LDL-C/HDL-C, TC/HDL-C, and TG/HDL-C were observed in PCOS rats. However, the intervention of AGC significantly reduced the mean value of ratios of LDL-C/HDL-C, TC/HDL-C, and TG/HDL-C compared to that of the PCOS group. No significant difference in ratios of LDL-C/HDL-C, TC/HDL-C, and TG/HDL-C mean value of control, AGC, and PCOS + AGC treatment groups (p > 0.05) [Table 5].

Ovarian oxidant and antioxidant parameters

The ovarian MDA levels of PCOS animals were significantly higher relative to the control (p < 0.05). Compared to the PCOS group, the mean value of ovarian MDA in the AGC and PCOS + AGC therapy groups decreased significantly (p < 0.5). In addition, the PCOS + AGC group had a somewhat higher value than the control and AGC groups (p > 0.05).

In comparison to the control group, the concentrations of SOD, CAT, and TP in the ovary decreased significantly (p < 0.05). Relative to the PCOS group, therapy with just AGC and after treatment with AGC significantly increased ovarian SOD, CAT, and TP concentrations (p < 0.05). [Fig. 6].

Fig. 6

Effects of Ageratumconyzoidesextracton ovarian follicle count in polycystic ovary syndrome rats. Data presented as mean ± S.E.M, n = 6 in each group, *: represent a significant difference from control, α: represent a significant difference from PCOS at p < 0.05, One-Way ANOVA. Follow by Tukey's multiple comparisons test, AGC: Ageratum conyzoides PCOS: Polycystic ovary syndrome.

Serum inflammatory markers

There was an observed significant increase in serum TNF-α, CRP, IL-6, and IL-18 in PCOS rats in comparison to control (p < 0.05). The administration of AGC alone and post-administration of AGC after PCOS induction significantly reduced the level of TNF-α, CRP, IL-6, and IL-18 relative to the PCOS group (p < 0.05). When compared to the control, AGC, and PCOS + AGC groups recorded no significant difference in TNF-α, CRP, IL-6, and IL-18 (p > 0.05).However, AGC presents an insignificant lower value in TNF-α, CRP, IL-6, and IL-18 than the control and PCOS + AGC (p > 0.05) [Table 7].

Table 7

Effect of Ageratum conyzoides extract on inflammatory markers of polycystic ovarian syndrome rat.

Parameters	Treatment groups
	Control	PCOS	AGC	PCOS + AGC
TNF-α (ng/g protein)	19.13 ± 4.94	48.01 ± 9.08*	16.31 ± 4.69α	23.09 ± 5.41α
CRP (ng/g protein)	19.53 ± 6.48	56.84 ± 13.62*	17.51 ± 6.01α	22.07 ± 6.82α
IL-6 (ng/g protein)	26.25 ± 8.66	60.40 ± 17.26*	23.50 ± 8.24 α	27.74 ± 8.79α
IL-18 (ng/g protein)	4.52 ± 1.61	15.33 ± 3.32*	3.73 ± 1.24α	5.85 ± 1.59α

Data presented as mean ± S.E.M, n = 6 in each group, *: represent a significant difference from control, α: represent a significant difference from PCOS at p < 0.05, One-Way ANOVA. Follow by Tukey's multiple comparisons test, AGC: Ageratum conyzoides PCOS: Polycystic ovary syndrome.

FBG, FINS, and HOMA-IR

Data on fasting blood glucose (FBG), fasting insulin (FINS), and HOMA-IR results are displayed in Fig. 4. FBG, FINS, and HOMA-IR levels were significantly increased in PCOS rats than in the control group (p < 0.05). In contrast to the PCOS group, rats in the AGC and PCOS + AGC therapy groups had significantly decreased FBG, FINS, and HOMA-IR values ( 0.05) [Fig. 4].

Fig. 4

Effects of Ageratum conyzoides extract on FBG, FINS, and HOMA-IR in Polycystic ovary syndrome rats. Data presented as mean ± S.E.M, n = 6 in each group, *: represent a significant difference from control, α: represents a significantly different from PCOS at p < 0.05, One-Way ANOVA. Follow by Tukey's multiple comparisons test, AGC: Ageratum conyzoides PCOS: Polycystic ovary syndrome.

Number of primary, secondary, and tertiary follicles

When comparing the PCOS group to the control group, the number of primary, secondary, and tertiary follicles decreased significantly (p < 0.05). In comparison to the PCOS group, post-administration of AGC following PCOS induction reversed this impact by substantially increasing the number of primary, secondary, and tertiary follicles (p < 0.05). In addition, as compared to the control, the number of primary follicles increased significantly in the AGS and PCOS + AGC therapy groups (p < 0.05). Between the AGC and PCOS + AGC experimental groups, no significant differences in primary, secondary, or tertiary follicles were recorded (p > 0.05) [Fig. 5].

Fig. 5

Effects of Ageratum conyzoides extract on the primary, secondary and tertiary follicular count in polycystic ovary syndrome rats. Data presented as mean ± S.E.M, n = 6 in each group, *: represent a significant difference from control, α: represent a significant difference from PCOS at p < 0.05, One-Way ANOVA. Follow by Tukey's multiple comparisons test, AGC: Ageratum conyzoides PCOS: Polycystic ovary syndrome.

Ovarian follicular count

The number of Graafian and corpora lutea follicles decreased significantly in the PCOS relative to the normal control group (p < 0.05). There was a significant increase in Graafian and corpora lutea follicles in AGC and PCOS + AGC groups compared to the PCOS group (p < 0.05) but the AGC group presented an insignificant higher value of the number of Graafian and corpora lutea follicles than the PCOS + AGC group (p > 0.05) [Fig. 6].

The number of cystic and atretic follicles increased significantly in the PCOS group as compared to the normal control group. Post-treatment with AGC reduced the number of cystic and atretic follicles significantly (p < 0.05) as compared with the PCOS group. Furthermore, treatment with only AGC exhibited an insignificant reduction in the number of cystic and atretic follicles as compared to the PCOS + AGC group [Fig. 6].

No cystic follicles were identified in the ovaries of normal control animals. However, in the ovaries of animals in the PCOS group, the number of cystic follicles was significantly higher compared to the control (p < 0.05). Notably, the number of cystic follicles decreased significantly following post-treatment with AGC, in comparison with the PCOS group. No significant difference was observed in the number of cystic follicles between AGC and PCOS + AGC treatment groups (p > 0.05) [Fig. 6].

Morphometry of antral follicles

When compared to the control group, the PCOS group had a significant increase (p < 0.05) in the diameter of antral follicles and the thickness of the theca layer. The thickness of the granulosa layer, on the other hand, decreased significantly (p < 0.05). In comparison to the PCOS group, the diameter of the antral follicle and theca layer reduced after treatment with AGC, whereas the diameter of the granulosa increased (p < 0.05). There was no significant change in theantral follicle diameter, theca layer, and granulosa diameter between the control, AGC, PCOS + AGC treated groups (p > 0.05) [Fig. 7].

Fig. 7

Effects of Ageratum conyzoides extract on morphometry of antral follicles in polycystic ovary syndrome rats. Data presented as mean ± S.E.M, n = 6 in each group, *: represent a significant difference from control, α: represent a significant difference from PCOS at p < 0.05, One-Way ANOVA. Follow by Tukey's multiple comparisons test, AGC: Ageratum conyzoides PCOS: Polycystic ovary syndrome.

Histomorphology of the ovary

Normal ovarian morphology with mature follicles (tertiary and Graafian follicles) and corpora lutea, which is a sign of ovulation, can be seen in photomicrographs of the ovarian sections of the normal control group [Fig. 8A].

Fig. 8

Photomicrographs of the ovarian cross-section. A: Control group displaying normal histological appearance with corpus luteum (CL) and several healthy follicles. B: Ovarian cross-sections of PCOS group showing many cystic follicles (CF) with lesser CL and healthy follicles. C: Ovarian Section of AGC group showing normal follicles with abundant CL and healthy follicles. D: Section of the PCOS + AGC group revealed improvement in the histological features with the absence of cystic follicles (CF) and the number of luteal bodies and follicles in different stages increased. Stained with [hematoxylin-eosin (H&E) Scale bar-50μm].

The presence of cystic and atretic follicles dominated the microarchitecture of the ovarian sections of PCOS animals (PCOS group) [Fig. 8B].

Normal ovarian morphology with mature follicles (tertiary and Graafian follicles) and corpora lutea, which is a sign of ovulation, can be seen in photomicrographs of the AGC group's ovarian sections [Fig. 8C].

The ovarian sections of the PCOS + AGC group revealed typical ovarian morphology, including mature follicles, corpora lutea, and a few cystic and atretic follicles [Fig. 8D].

Histomorphology of the uterus

Photomicrographs of uterine sections from the control group featured normal morphology with a tall cuboidal epithelial outline [Fig. 9A].

Fig. 9

Photomicrograph of A: control showed normal simple cuboidal epithelial cells lining the uterine gland, and well-pigmented endometrial cells, B: PCOS revealed severe uterine cellular distortion, C: AGC, shows normal histological appearance with normal endometrium lining (EL), and uterine gland (UG) D: PCOS + AGC displaying an improvement in the histological structures with visible endometrium lining (EL), thick uterus and uterine gland almost comparable with the control group. Stained with [Hematoxylin & eosin, Scale bar- 50 μm]. E; Epithelium, L: lumen, UG: uterine glands, EL: Endometrial lining.

Photomicrographs of uterus sections from PCOS rats showed degenerated uterine tissue bordered by a poor cuboidal epithelium [Fig. 9B].

Normal morphology with a tall cuboidal epithelial shape may be seen in photomicrographs of uterine sections from the AGC group [Fig. 9C].

Photomicrographs of uterus sections from the PCOS + AGC group indicate changes in uterine epithelial cell shape, with uterine epithelial cells becoming cylindrical and uterine epithelial cells hypertrophy [Fig. 9D].

Uterine histomorphometric parameters

Morphometric measurements in a section of the uterus of the PCOS rats showed a significant reduction in epithelial height, length of the uterine, endometrium thickness, and numbers of the endometrial gland respectively compared to the control group (p < 0.05). However, AGC and PCOS + AGC experimental groups displayed a significant increase (p < 0.05) in epithelial height, length of the uterine, endometrium thickness, and numbers of the endometrial gland compared to the PCOS group. The mean value of epithelial height, length of the uterine, endometrium thickness, and numbers of the endometrial gland in AGC and PCOS + AGC treatment groups showed no significant difference (p > 0.05) [Fig. 10].

Fig. 10

Effects of Ageratum conyzoides extract on uterine morphometry parameters in polycystic ovary syndrome rats. Data presented as mean ± S.E.M, n = 6 in each group, *: represent a significant difference from control, α: represents a significantly different from PCOS at p < 0.05, One-Way ANOVA. Follow by Tukey's multiple comparisons test, AGC: Ageratum conyzoides PCOS: Polycystic ovary syndrome.

There was observed a significantly higher value in the numbers of eosinophil cells in the uterine stroma in PCOS rats as compared to the control. But, reduced significantly after treatment with AGC compared to the PCOS group. The group treated with AGC alone also shows a significant reduction in the numbers of eosinophil cells compared to the PCOS group (p < 0.05) [Fig. 10].

Discussion

In this study, the estrous cycle was disrupted as a result of the EV injection, which increased body weight, BMI, and Lee's index. Throughout the trial period, Lee's index (LI) values for the PCOS group were greater than 310, demonstrating the effectiveness of EV in the induction of PCOS. The significant difference between the initial and final BMI in PCOS rats corroborated the findings of Dadachanji et al. [65], Neubronner et al. [66], and Wang et al. [67].

Ageratum conyzoides intervention reduced the mean BMI and LI, corroborating reports that a little weight loss of about 5% can improve insulin resistance, hormone levels, menstrual cycles, and infertility in women with PCOS [68]. Also, in the AGC and PCOS + AGC therapy groups, there was a decrease in HOMA-IR and an improvement in estrous cyclicity, validating AGC's insulin-sensitizing ability. Marvel et al. [69] and Dou et al. [70] both reported that insulin sensitizers may enhance menstrual cyclicity and ovulation in PCOS patients. Obesity and abdominal obesity aggravate the symptoms of menstruation irregularity and infertility, and are linked to higher levels of androgens and luteinizing hormones in the blood [71]. In both women with PCOS and controls, an increase in body weight is linked to an increase in androgen levels [72]. In the etiology and pathophysiology of PCOS, a complicated connection occurs between obesity, abdominal obesity, insulin resistance, androgen level, and LH level [73]. Elevated body weight and the presence of estrous cycle anomalies in rats following oral administration of EV injection imply the development of PCOS in rats due to increased androgen and LH hormone levels in our study. When compared to the PCOS group, the AGC and PCOS + AGC therapy groups showed a considerable reduction in body weight. After induction of PCOS, treatment with AGC decreased fat buildup and increased ovarian and uterine weight. Also, AGC caused the estrous cycle to resume after it had been stopped in the diestrus phase following EV injection. This might be connected to lower blood testosterone and LH levels and higher estradiol levels, which would benefit ovarian follicular growth and maturation, follicle atresia reduction, and corpora lutea formation and maintenance. We may assume that the bioactive component of AGC has overcome the inhibitory effect of EV, resulting in lower levels of testosterone and LH and higher levels of progesterone and estradiol. The progesterone and estradiol would have accelerated the proliferation of uterine epithelial cells. This uterotrophic effect is known to be mediated by estrogen receptor alpha [74]. Furthermore, because AGC reduced circulating free androgen, the improvement in ovarian dynamic indicated that AGC reduced androgen stimulation of insulin release by pancreatic cells. Hyperinsulinemia caused by hyperandrogenism has been linked to early granulosa cell maturation and luteinization, follicular growth arrest, anovulation, and cyst development [75]. Low levels of high-density lipoprotein cholesterol (HDL-C), high levels of triglycerides, total cholesterol, and low-density lipoprotein cholesterol (LDL-C) are all common symptoms of the polycystic ovarian syndrome [76].

Although extensive series of data shows that the mean circulating lipid levels in women with PCOS are within acceptable limits, up to 70% of individuals have at least one aberrant lipid level [77].

In our study, the levels of glucose, cholesterol, and triglyceride levels in PCOS rats were all considerably higher. However, after treatment with AGC, the levels of glucose, cholesterol, and triglycerides all decreased dramatically. In PCOS rats, there was a considerable rise in LDL-C and VLDL-C. However, in the PCOS group, HDL-C levels are considerably lower than in the control group. The activation of the hormone-sensitive lipase in PCOS rats was mostly due to an increase in the release of fatty acids from peripheral tissue to the blood vessels, which might be attributable to an increase in the release of fatty acids from peripheral tissue to the blood vessels. As a result, treatment with AGC following PCOS induction raises HDL-C while lowering VLDL-C and HDL-C. The considerable fall in glucose levels following AGC administration might be due in part to the bioactive components of AGC promoting glucose entrance into cells, hence reducing glucose release into the bloodstream. Finally, it appears that AGC extracts affect the metabolic complication of PCOS (dyslipidemia). Carani et al. [78] and Orostica et al. [79] reported that inflammation is activated and increased in different endometrial cells and the serum of PCOS patients, and the current study shows that the expression of an array of inflammation-related markers is dramatically altered in the PCOS-like rat uterus. TNF and IL-6 levels in the uterus are enhanced in PCOS-like mice, indicating that TNF and IL-6 have an inflammatory role in endometrial function in PCOS patients [80]. Low-grade inflammation has been linked to PCOS [81], which supports the current findings, which reveal that serum IL-6, IL-18, TNF-, and CRP levels are considerably higher in PCOS rats. The anti-inflammatory activity of AGC, which improved the low-grade inflammation state and downregulated pro-inflammatory cytokine production (TNF-α, IL-18, and IL-6) in this study, might explain why AGC intervention dramatically reduced the levels of inflammation components in PCOS rats. The imbalance between oxidants and antioxidants, as well as the increased creation of reactive oxygen species (ROS), is referred to as oxidative stress [82,83]. In PCOS, the production of reactive oxygen species (ROS) by mononuclear cells (MNCs) in response to hyperglycemia increased, regardless of weight [84]. In PCOS, oxidative indicators in the bloodstream are much higher, and oxidative stress plays a key role in PCOS pathogenesis [85].

In this study, ovarian SOD, CAT, and total peroxidase concentrations were much lower in PCOS rats, whereas ovarian MDA was significantly greater in PCOS rats, which was consistent with earlier research on PCOS patients [86,87]. In this paradigm, oxidative stress was verified in PCOS rats. In comparison to control rats, post-treatment with AGC dramatically increased ovarian SOD, CAT, and total peroxidase and decreased ovarian MDA. This might be related to AGC's antioxidant ability, which mops up free radicals and reduces ROS. As a result, we may conclude that AGC is efficient in reducing oxidative stress and improving the antioxidant system in the PCOS rat model.

Endometrial deficiencies may be the cause of implantation failure in PCOS patients, as evidenced by dysregulation of the expression of proteins necessary for implantation in the human endometrium [88]. AGC therapy restores endocrine (hyperandrogenism), metabolic (insulin resistance), and reproductive (lack of estrous cycle) problems, as well as partially reverses implantation failure in most PCOS-like rats, according to our findings. It has been discovered that several chemicals in the uterus are essential for implantation and that mutations in these molecules result in infertility [89]. In addition, investigations have shown that in PCOS individuals, endometrial decidualization is hindered [90].

In this study, histological studies of uterine tissue sections of PCOS rats revealed a considerable increase in uterine wall thickness and epithelial height as compared to the control group. It has been demonstrated that estrogen-mediated uterine stimulation causes morphogenetic changes, such as changes in the kind and shape of luminal and glandular epithelia [91]. In vitrostudies of radiothymidine uptake by endometrium suggest that high levels of estradiol are primarily associated with maximal proliferation in uterine glands and stroma [92], and that ovarian steroids are among the most important factors that affect uterine morphology and motility [93,94].

The findings of this study also backed up the theory that an aberration in ovarian steroids causes obvious alterations in the epithelial surface, gland accumulation, and overall thickness of the uterine wall. In addition, an increase in eosinophil quality in the endometrial stroma in PCOS rats was detected in the current study, confirming a valuable report that PCOS increases eosinophil quality in the uterine stroma [95]. The influence of hormonal changes on reproductive tissues revealed that leukocyte entry into uterine tissues is mostly controlled by hormones [96]. PCOS has been shown to cause histo-architectural abnormalities in the ovary [97] and uterine of rats [98].This study found that PCOS rats have the leastcorpus luteum and numerous follicular ovarian cysts, which is consistent with animal models of PCOS [99,100]. Reduced levels of testosterone, FSH, LH, and antioxidant activity might be linked to decreased ovarian weight, uterine length and weight, endometrium thickness, and endometrial gland numbers. In the AGC and PCOS + AGC therapy groups, there wereincrease in corpora lutea numbers, ovarian and uterine weight, endometrium thickness, and endometrial gland numbers, as well as a decrease in cystic follicle counts. The presence of corpora lutea indicates that the animals have ovulated [101].

Higher levels of progesterone in AGC post-treated rats and rats treated with AGC alone indicated the occurrence of ovulation, indicating that AGC had restored ovulation in the PCOS rats. This is significant because the primary goal of PCOS care is to restore ovulation. The favorable effect of AGC on the histomorphology and histomorphometry of the uterus and ovaries might be due to the high antioxidant, anti-inflammatory, and pro-fertility effects of the bioactive component of AGC. Treatment with AGC resulted in substantial improvements in testosterone, estrogen, LH levels, and ovarian and uterine tissue. The histological findings in the AGC-treated group revealed a significant recovery of ovarian tissue, with the existence of several follicles in various stages of development, indicating normal oogenesis. The granulosa layer was normal, with distinct thecal layers in the follicles. The existence of corpora lutea following AGC therapy demonstrated that AGC treatment restored the normal estrous cycle. AGC had an anti-androgenic effect, lowering androgen levels and preventing ovarian cell malfunction in PCOS patients, resulting in improved fertility [102]. AGC's success in the treatment of PCOS may be due to the observed healing of ovarian tissue as well as its antiandrogen capability. This study has several limitations. First, the levels of TNF-α, IL-6 and IL-18 expression were not identified. Second, ovarian cell count and uterine histomorphometric took several weeks to establish and were very time-consuming. Third, leptin and Anti-Müllerian hormone were not verified in this study. In the next step, metformin or other insulin sensitizers and combination of metformin andAgeratum conyzoides will be used to treat PCOS rats to verify the changes of the insulin signaling pathway or to assess new therapeutic methods in this model.

Conclusion

Infertility is a serious worry for PCOS patients, and follicular atresia, anovulation, and subsequent hyperandrogenemia are common causes of infertility. Phytotherapy is gaining popularity due to perceived low cost, availability, efficacy and few side effects. This is more prominent in low-income countries of the world. Ageratum conyzoides can be utilized to treat infertile individuals whose infertility is caused by low oocyte quality and anovulation. We find that Ageratum conyzoides can improve insulin sensitivity, lipid profile, antioxidant activity, hormonal balance, and anti-inflammatory activity in female rats, thus reversing the symptoms of PCOS. These findings add to our existing knowledge of the possible use of Ageratum conyzoides as an antioxidant for the treatment of PCOS and as an alternative therapy for the treatment of PCOS.

Funding

This study got no particular support from the government, commercial, or non-profit funding entities. It was carried out as part of the authors' obligation and condition of services at the Animal Facility of the School of Basic Medical Science, Federal University of Technology, Akure, Nigeria.

Availability of data and materials

All data generated or analyzed during this study are included in this manuscript.

CRediT authorship contribution statement

Sunday Aderemi Adelakun: Conceptualization, Methodology, Validation, Writing – review & editing, Investigation, Writing – original draft. Victor Okoliko Ukwenya: Formal analysis, Investigation, Writing – original draft. Akwu Bala Peter: Methodology, Project administration, Supervision, Investigation. Adewale Jacob Siyanbade: Writing – original draft, Investigation. Comfort Oluwakorede Akinwumiju: Writing – original draft, Investigation.

Declaration of competing interest

The authors declare that they have no competing interests.