Thyme Extract Regulates Ovarian Gene Expression in Experimental Polycystic Ovary Syndrome

In a controlled laboratory investigation using a letrozole-induced polycystic ovary syndrome rat model, an ethanolic extract from Thymus vulgaris leaves administered orally for fourteen consecutive days produced a dose-dependent reduction in ovarian gene expression levels of inhibin A, inhibin B, and aromatase (CYP19A1), while restoring hormonal balance, ovarian histoarchitecture, and follicular development. Researchers observed that treatment with the lowest evaluated botanical dose of 50 mg/kg significantly suppressed ovarian messenger RNA levels of inhibin A by 90 percent, inhibin B by 87 percent, and CYP19A1 by 77 percent, closely mirroring or exceeding the therapeutic benefits of the standard insulin-sensitizing medication metformin. Polycystic ovary syndrome, an endocrine disorder originally described as Stein–Leventhal syndrome that affects females of reproductive age, represents a leading cause of female anovulatory infertility and hyperandrogenism, driven by complex disruptions across the hypothalamic-pituitary-ovarian regulatory axis.

Under the diagnostic criteria established by the Rotterdam consensus, polycystic ovary syndrome is diagnosed by the presence of at least two clinical features among oligomenorrhea, ultrasound evidence showing multiple sub-10-millimeter ovarian follicles arrayed in a classic pearl necklace pattern beneath the ovarian capsule, and hyperandrogenism featuring irregular menstrual cycles, hirsutism, acne, and infertility. Although insulin resistance is not formally included in the Rotterdam criteria, it represents a pervasive feature in affected females requiring clinical consideration due to its established link to long-term cardiovascular risks and metabolic disturbances. Hormonal imbalances in the syndrome—specifically hyperandrogenism combined with hyperinsulinemia—enhance adipogenesis by stimulating preadipocyte differentiation and accelerated lipid accumulation in adipose depots. Current pharmacological management relies predominantly on insulin sensitizers like metformin, combined oral contraceptives, anti-androgens, and ovulation-inducing medications to regulate cycles and mitigate androgen excess, yet these conventional therapies frequently present limitations, including short-term efficacy and clinical adverse effects.

At the molecular foundation of ovarian regulatory signaling, inhibins function as dimeric glycoproteins within the transforming growth factor-beta superfamily that orchestrate mammalian reproductive processes. These bioactive glycoproteins are disulfide-linked heterodimers assembled from an alpha subunit linked to either a beta-A subunit yielding inhibin A or a beta-B subunit forming inhibin B, secreted predominantly by follicular granulosa and theca cells. Circulating concentrations fluctuate across the menstrual cycle, with inhibin A peaking during mid-follicular and mid-luteal phases, while inhibin B peaks during the early follicular stage. Functionally, inhibins exert critical negative feedback suppression on the anterior pituitary gland to modulate follicle-stimulating hormone secretion, an essential driver of follicular maturation and estrogen biosynthesis. In polycystic ovary syndrome, elevated circulating inhibin A and inhibin B create an aberrant, premature negative feedback loop that suppresses pituitary follicle-stimulating hormone release, driving follicular arrest. Concurrently, aromatase, encoded by CYP19A1, catalyzes the conversion of C19 androgens into C18 estrogens, a process disrupted in polycystic ovaries.

To investigate these pathological mechanisms, researchers utilized adult female Wistar strain albino rats obtained from the National Research Center animal facility in Cairo, Egypt. A priori statistical power calculations conducted using G*Power software determined that an experimental cohort of sixty-three rats, aged six weeks and weighing 150 to 250 grams, divided into groups of seven animals each, provided a statistical power of 0.8 with an alpha error probability of 0.05 and an effect size of 0.57. Aerial parts of Thymus vulgaris were collected in January 2019 from the Central Botanical Garden at the National Research Centre in Giza, Egypt, verified taxonomically, and deposited under voucher specimen PHBL00412 at the German University in Cairo. Three kilograms of air-dried leaves were exhaustively extracted by cold maceration in 75 percent aqueous ethanol across three cycles of three days each, followed by rotary evaporation at 50 to 60 degrees Celsius to yield 180 grams of semisolid brown extract.

Experimental polycystic ovary syndrome was induced by administering letrozole, a nonsteroidal aromatase inhibitor, dissolved in 0.5 percent carboxymethylcellulose orally at a dosage of 1 mg/kg daily for 21 consecutive days. Successful syndrome induction was verified by microscopic evaluation of saline-moistened, methanol-fixed vaginal smears to detect estrous cycle arrest and persistent anovulation. Animals were assigned to two primary cohorts: Group I, consisting of non-PCOS controls, and Group II, comprising letrozole-induced PCOS animals. Following induction confirmation, diseased rats received daily oral treatments for 14 consecutive days consisting of either 300 mg/kg metformin or total Thymus vulgaris extract at doses of 50, 75, or 100 mg/kg. Phytometabolite profiling of the extract was performed using HPLC-PDA-ESI-MS/MS. Serum endocrine and metabolic parameters were quantified via microplate ELISA readers, while excised right ovaries underwent total RNA extraction, spectrophotometric purity evaluation, reverse transcription, and real-time quantitative PCR amplification using QuantiTect primers targeting CYP19A1, inhibin A, and inhibin B.

Assessment of anatomical and systemic metabolic parameters revealed that letrozole induction provoked substantial physiological derangements compared to vehicle-treated control animals. Induction of polycystic ovary syndrome markedly increased total body weight by 38.9 percent and reproductive organ weight by 44.5 percent relative to healthy controls. Oral administration of Thymus vulgaris extract significantly reversed this reproductive organ enlargement across all tested dose tiers: reproductive organ weights in PCOS rats treated with 50, 75, and 100 mg/kg thyme extract decreased by 38.3 percent, 34.1 percent, and 36.6 percent, respectively, compared to untreated letrozole rats, closely paralleling the 36.6 percent reduction recorded under 300 mg/kg metformin therapy. Furthermore, letrozole exposure prompted a 36.56 percent increase in fasting blood glucose levels over negative control baselines. Therapeutic intervention with Thymus vulgaris extract produced notable metabolic improvements: serum fasting blood glucose concentrations decreased by 27.03 percent in the 50 mg/kg thyme group, 17.52 percent in the 75 mg/kg group, and 26.27 percent in the 100 mg/kg group, whereas metformin achieved a 25.5 percent decrease.

Endocrine evaluations demonstrated that letrozole intoxication caused profound steroidogenic disruptions, escalating circulating serum testosterone concentrations by 249.97 percent while depleting estrogen and progesterone levels by 67.53 percent and 92.33 percent, respectively, relative to control rats. Treatment with Thymus vulgaris leaf extract substantially corrected these pathological hormonal imbalances. Circulating testosterone levels decreased by 88.02 percent in the 50 mg/kg thyme group, 61.24 percent in the 75 mg/kg group, and 73.11 percent in the 100 mg/kg group, compared to a 94.32 percent reduction induced by metformin. Notably, the 50 mg/kg thyme dose achieved a significantly greater reduction in testosterone than higher extract doses, exhibiting efficacy comparable to metformin. Concurrently, serum estrogen concentrations increased by 265.52 percent, 315.29 percent, and 245.04 percent under 50, 75, and 100 mg/kg thyme extracts, respectively, compared to 209.07 percent with metformin. Most prominently, thyme extract stimulated remarkable surges in serum progesterone, which elevated by 1326.64 percent, 1089.21 percent, and 1384.70 percent across the three extract groups, significantly surpassing metformin, which produced no significant progesterone increase.

The study also revealed substantial corrections in circulating gonadotropins and ovarian molecular targets. Letrozole treatment reduced serum follicle-stimulating hormone by 73.33 percent while increasing luteinizing hormone by 71.4 percent, producing a twofold expansion of the luteinizing hormone to follicle-stimulating hormone ratio. Co-administration of Thymus vulgaris extract counteracted this disturbance, raising follicle-stimulating hormone levels by 150.05 percent, 163.85 percent, and 183.29 percent at 50, 75, and 100 mg/kg, respectively, while lowering luteinizing hormone concentrations by 46.65 percent, 51.65 percent, and 48.32 percent, effectively reducing the ratio by 52.29 percent, 51.02 percent, and 61.07 percent. At the transcriptional level, letrozole elevated ovarian gene expression of inhibin A by 115 percent, inhibin B by 880 percent, and CYP19A1 by 2800 percent. The 50 mg/kg thyme extract suppressed inhibin A mRNA expression by 90 percent, inhibin B by 87 percent, and CYP19A1 by 77 percent. Higher extract doses reduced inhibin A by approximately 47 percent and inhibin B by 78 percent, but exerted no significant change on CYP19A1 expression. Immunohistochemistry confirmed mild inhibin presence across thyme-treated cohorts compared to severe staining in untreated PCOS rats.

Histopathological examination of ovarian tissue sections stained with hematoxylin and eosin provided physical confirmation of these endocrine and molecular changes. Ovaries from negative control animals exhibited normal histological architecture featuring various stages of follicular maturation, mature Graafian follicles, and healthy interstitial stromal cells. In contrast, untreated letrozole-induced PCOS ovaries displayed marked inflammatory infiltration in the ovarian stroma, vascular congestion, and multiple follicular cysts reflecting anovulatory arrest. Treatment with metformin produced partial histological recovery, presenting few primordial, primary, and secondary follicles, but lacking mature Graafian follicles. Conversely, ovaries from rats receiving 50 mg/kg Thymus vulgaris extract displayed restoration of ovarian histoarchitecture characterized by follicular maturation, mature Graafian follicles, and healthy corpora lutea indicating successful ovulation, although cyst formation reappeared at doses of 75 and 100 mg/kg. Phytochemical profiling by liquid chromatography-mass spectrometry identified diverse bioactive metabolites, including flavonoids, phenolic acids, and glycosides such as apigenin, quercetin, luteolin, dihydroquercetin derivatives, and p-coumaroyl quinic acid, compounds documented to exert antiandrogenic activity by modulating steroidogenesis and regulating aromatase transcription.

The biological finding that the lowest evaluated botanical dose of 50 mg/kg conferred superior therapeutic modulation over higher concentrations indicates a potential biphasic response or hormetic dynamic, wherein lower and higher doses elicit divergent biological outcomes. The authors noted that higher botanical concentrations may reach biological saturation thresholds or stimulate compensatory metabolic clearance pathways that diminish extract efficacy, necessitating additional investigations to pinpoint the precise molecular constituents responsible for this dosage profile. Translating these preclinical outcomes, the researchers calculated an equivalent human clinical dose of 8 mg/kg based on the effective 50 mg/kg animal dose using body surface area normalization methodology. However, the study represents a preclinical rodent trial carried out in a chemically induced model over a fourteen-day experimental timeframe, and these findings cannot be interpreted as established human clinical efficacy. The authors concluded that while Thymus vulgaris demonstrated multifaceted preclinical potential in regulating inhibin A, inhibin B, and CYP19A1 expression while restoring ovarian architecture and metabolic markers, controlled human clinical trials remain necessary to evaluate its safety, pharmacokinetics, and therapeutic utility before it can be recommended as a clinical intervention for polycystic ovary syndrome.

Subject of Research: Evaluation of Thymus vulgaris leaf extract on ovarian gene expression and endocrine balance in a letrozole-induced PCOS rat model

Article Title: Potential effects of Thymus vulgaris L. on the gene expression of inhibin A, inhibin B, and Aromatase (CYP19A1) in a letrozole-induced PCOS rat model

Article References: Sabra, H., El-Zeiny, N., Atwa, S., Rady, M., Maksoud, S. A., & Handoussa, H. (2026). Potential effects of Thymus vulgaris L. on the gene expression of inhibin A, inhibin B, and Aromatase (CYP19A1) in a letrozole-induced PCOS rat model. Future Journal of Pharmaceutical Sciences, 12(1), Article 75. https://doi.org/10.1186/s43094-026-00995-8

Image Credits: AI Generated

DOI: 10.1186/s43094-026-00995-8

Keywords: Polycystic Ovary Syndrome, Thymus vulgaris, Inhibin A, Inhibin B, CYP19A1, Aromatase, Letrozole, Metformin, Hormonal Imbalance, Preclinical Pharmacology, Potential, effects