Web of Science, Embase, Scopus, Cochrane, and PubMed databases were systematically searched for relevant studies published in English, between January 2000 and February 2024, without any time limitation to retrieve experimental, clinical trials, animal studies, and cell studies that examined the GABAergic pathway implicated in PCOS, and assessed the impact of GABA on this syndrome.

The search strategies used were consistent across all databases and were performed using MeSH terms, focusing on titles, abstracts, and keywords: (PCOS OR “Polycystic Ovary Syndrome” OR “Ovary Syndrome, Polycystic” OR “Syndrome, Polycystic Ovary” OR “Stein-Leventhal Syndrome” OR “Stein Leventhal Syndrome” OR “Syndrome, Stein-Leventhal” OR “Sclerocystic Ovarian Degeneration” OR “Ovarian Degeneration, Sclerocystic” OR “Sclerocystic Ovary Syndrome” OR “Polycystic Ovarian Syndrome” OR “Ovarian Syndrome, Polycystic” OR “Polycystic Ovary Syndrome” OR “Sclerocystic Ovaries” OR “Ovary, Sclerocystic” OR “Sclerocystic Ovary”) AND (“γ-Aminobutyric acid” OR GABA OR “gamma Aminobutyric Acid” OR “Aminalon” OR “Aminalone” OR “4-Aminobutanoic Acid” OR “4 Aminobutanoic Acid” OR “4-Aminobutyric Acid” OR “4 Aminobutyric Acid” OR “gamma-Aminobutyric Acid”, Hydrochloride OR “Acid, Hydrochloride gamma- Aminobutyric” OR “Hydrochloride gamma-Aminobutyric Acid” OR “gamma Aminobutyric Acid”, Hydrochloride OR Gammalon OR “gamma-Aminobutyric Acid”, “Monosodium Salt” OR “gamma Aminobutyric Acid”, “Monosodium Salt” OR “gamma-Aminobutyric Acid”, “Zinc Salt” OR “gamma- Aminobutyric Acid”, “Calcium Salt” OR “gamma-Aminobutyric Acid”, “Monolithium Salt” OR “gamma Aminobutyric Acid”, “Monolithium Salt” OR “Lithium GABA” OR “GABA, Lithium”).

The searches were conducted based on titles, abstracts, and keywords. Based on the ‘pearl growing’ strategy, after obtaining the full-text articles, the reference list of all included studies was reviewed for any additional publications that could be used in this review. Studies with other designs, such as reviews, case reports, case series, guidelines, commentary, abstracts and articles with unreliable and incomplete results, and studies that did not assess both outcomes of interest were excluded.

Two reviewers independently screened the titles and abstracts, followed by a full-text review to determine study eligibility. All searches were conducted by two reviewers (F.M and F.RT). The search results were screened based on predefined eligibility criteria. All references were entered into EndNote software. The initial selection was based on the title. Duplicates were deleted, and all remaining abstracts were reviewed. The full texts of all selected articles were obtained for data processing. Disagreements were resolved through discussion and, if necessary, consultation with a third reviewer.

The methodological quality of the included studies was assessed using the Cochrane Collaboration’s tool to assess the risk of bias and the Newcastle-Ottawa scale (NOS). The Cochrane Collaboration’s tool examines various domains of potential bias, including selection bias, performance bias, detection bias, attrition bias, reporting bias, and other sources of bias. Each included study was independently assessed by two reviewers, who evaluated the risk of bias as “low”, “high”, or “unclear” for each domain. Discrepancies between the reviewers’ assessments were resolved through discussion. The risk of bias was categorized as low, moderate, or high based on specific criteria. For the NOS, three domains were scored for selection and comparability of study groups, and to determine the outcome of interest. If a study obtained ≥70% of the highest level of the NOS, it was considered as high quality, those with 40-70% as moderate, those with 20-40% as low, and those with <20% as very low quality.

Prenatal exposure to dihydrotestosterone (DHT), a potent androgen, disrupts the GABA-GnRH network in the developing fetal brain via androgen receptors, resulting in an abnormal estrous cycle, obesity, and anovulation in adulthood [13-15]. GABAergic neurons in the arcuate nucleus of the hypothalamus modulate LH secretion [16-18], indicating their involvement in regulation of the reproductive cycle [19]. Morphological evidence suggests that microglia play a role in the excessive GABAergic connectivity that leads to PCOS-like features in the prenatal androgenized brain. These findings suggest that microglia may contribute to the development or progression of PCOS-like conditions by influencing brain structure and function [20]. In a mouse model of PAE-induced PCOS, the expression of progesterone receptor protein was decreased in the arcuate nucleus of GABAergic neurons [15]. Furthermore, alterations in the neuropeptide Y/agouti-related peptide/GABA circuit that interacts with GnRH neurons suggest impairments in the regulation of reproductive hormones and the hypothalamic- pituitary-gonadal (HPG) axis [21]. PAE mice exhibit reduced GnRH neuron activity during prepuberty, leading to an increased firing rate of these neurons in adulthood [22]. GABAergic neurons in the anteroventral periventricular nucleus can activate the HPG axis and induce PCOS-like reproductive deficits in healthy females [17,23]. Anti mullerian hormone affects GnRH neuronal electrical activity and hormone release. Chronic inhibition of androgen receptor signaling from early adulthood restores normal GABAergic innervation of GnRH neurons, improves ovarian morphology, and rescues the reproductive cycle in PAE mice. Enhanced numbers of excitatory GABAergic inputs to the medial basal hypothalamic GnRH and kisspeptin/neurokinin B/dynorphin neurons following prenatal testosterone exposure may contribute to disturbances in steroid feedback mechanisms and elevated GnRH/LH plasticity [24-28] (Table 1).

In the PCOS animal model, dysregulation of the expression of hypothalamic neuropeptides has been well-documented. GnRH, the primary regulator of the HPG axis, plays a crucial role in reproductive functions. Kisspeptin and RF-amide related peptide-3 (RFRP-3) are important modulators of GnRH secretion, whereas ghrelin is implicated in the regulation of energy homeostasis and metabolic processes [29,30].

Various experimental studies on the GABAergic pathway have been conducted to restore the altered expression patterns of key hypothalamic neuropeptides in animal models [31,32].

These studies suggest that the targeted modulation of gene expression profiles within the hypothalamus may represent a potential therapeutic approach for addressing the complex hormonal and metabolic disturbances associated with PCOS.

Baclofen reversed alterations in gene expression of GnRH, kisspeptin, RFRP-3, and ghrelin in the hypothalamus of female rats with PCOS. It restores the equilibrium between the excitatory and inhibitory factors that control GnRH and LH secretion [6]. Troxerutin attenuated body weight gain and ovarian damage induced by DHT. It lowered the elevated levels of GnRH, LH, FSH, and testosterone in PCOS rats. Troxerutin normalized the expression of kisspeptin and neurokinin B in GnRH-positive neurons in the median eminence. It regulated the levels of GABA and glutamate in the hypothalamus, which influence GnRH release [33]. GABA ameliorated metabolic and reproductive disturbances in letrozole-induced PCOS rats. GABA significantly decreased body weight, body mass index, and testosterone level. GABA increased the levels of catalase, superoxide dismutase, peroxidase, and estradiol. GABA improved the lipid profile, glucose tolerance, and oral glucose tolerance test. GABA reduced the number of cystic follicles and adipocytes in the ovaries, enhancing ovarian morphology and function. GABA treatment may have therapeutic potential for treating PCOS in humans [34]. Allopregnanolone enhanced the amplitude and duration of GABAergic inhibition in GnRH neurons. Sullivan and Moenter [35] investigated the specific effects of dehydroepiandrosterone sulfate (DHEAS) on the electrophysiological properties of GABA receptor-mediated chloride currents. The results demonstrated that DHEAS reduced the amplitude of GABA receptor-mediated currents, but did not significantly affect the decay time of these currents. Importantly, the observed effects of DHEAS on GABA receptor currents were dose dependent, with modulation occurring within a physiologically relevant concentration range of 0.1 to 10 micromoles. This dose-dependent relationship suggests that the neurosteroid DHEAS can exert differential and concentration-specific effects on the function of GABA receptors [35] (Table 2).

GABA is a ubiquitous neurotransmitter in the central nervous system that plays a crucial role in the regulation of various physiological functions, including mood, anxiety, sleep, and hormone regulation. Several studies have investigated the potential links between GABA and PCOS, exploring the role of the GABAergic pathway in females with PCOS. These studies can be broadly categorized into two subgroups: 1) studies reporting GABA dysfunction as a consequence of PCOS and 2) studies proposing that PCOS-related features may arise as a result of primary GABA dysfunction. This categorization was based on the directionality of the proposed relationship between GABAergic signaling and the development of PCOS-associated characteristics. The first subgroup encompasses studies that have identified alterations in GABA neurotransmission as a secondary consequence of the hormonal imbalances and metabolic disturbances that are characteristic of PCOS. The second subgroup included investigations that hypothesized that primary disruptions in GABAergic pathways may contribute to the manifestation of PCOS-related features, such as reproductive dysfunction and metabolic abnormalities.

Several studies have reported alterations in GABA neurotransmission secondary to the hormonal and metabolic disturbances associated with PCOS. Liang et al. [36] showed that GABA-producing species (Parabacteroides distasonis, Bacteroides fragilis, and Escherichia coli) were increased in females with PCOS, and their levels were positively correlated with the serum concentration of LH and the LH: FSH ratio. Several studies have examined the potential relationship between PCOS and various psychiatric disorders that may result from GABA dysfunction. A recent study by Jin et al. [37] investigating this association in European populations reported that PCOS may be a causal factor in the development of obsessive-compulsive disorder, but not anxiety disorder, bipolar disorder, major depression disorder, or schizophrenia.

Aydogan Kirmizi et al. [38] investigated the relationship between PCOS and depression and reported that individuals with PCOS had significantly higher levels of inflammatory markers compared to healthy controls. Patients with PCOS also presented with elevated levels of depression-related biomarkers relative to the control group. Moreover, the severity of depressive symptoms in patients was found to be associated with lower concentrations of GABA and the neurotrophin brain-derived neurotrophic factor. Conversely, the severity of depression in PCOS was linked to a higher level of the excitatory neurotransmitter glutamate and increased inflammation markers [38]. Another study by Radwan et al. [10] suggested that a disrupted GABA level in the peripheral circulation is an additional contributing factor to the manifestation of PCOS. They found that GABA deficiency correlated with 25-hydroxyvitamin D₃ vitamin D deficiency, dyslipidemia, and total testosterone [39]. Higher cerebrospinal fluid levels of GABA and estradiol, and possibly T, than eumenorrheic, ovulatory women were also observed in women with PCOS [40].

Several studies have proposed that GABA dysfunction contributes to the manifestation of PCOS-related features, directly or indirectly. Ye et al. [41] investigated amino acid metabolism in females with PCOS and reported that these patietns had severe dysregulation of amino acid metabolism compared with healthy controls. They found that a combination of specific amino acids (AAs), including alanine, valine, leucine, tyrosine, glutamic acid, cysteine, and glycine, demonstrated potential predictive value for assessing the risk of metabolic syndrome in females with PCOS. Moreover, elevated levels of branched-chain AAs, tyrosine, alanine, and lysine were positively correlated with insulin resistance in the PCOS cohort. Alterations in the concentrations of tyrosine, lysine, methionine, hydroxyarginine, 3-methylhistidine, GABA, methyl histidine, and glycine were associated with obesity in females with PCOS [41]. Abnormal central GABA signaling has been identified in patients and in preclinical models as a possible link between androgen excess and elevated GnRH/LH secretion [42]. Females with PCOS had an elevated baseline allopregnanolone concentration compared with follicular-phase controls; however, the mediating effect of obesity on this association is not clear; obese females, regardless of PCOS status, were less sensitive to allopregnanolone than normal-weight controls [43] (Table 3).

To advance our understanding of the GABAergic pathway in PCOS, future studies should prioritize well-designed randomized controlled trials with larger sample sizes to evaluate the efficacy and safety of GABA supplementation across diverse PCOS phenotypes, utilizing standardized GABA formulations and dosages. These trials should incorporate placebo controls, blinding, and comprehensive monitoring of endocrine, metabolic, reproductive, and quality of life outcomes. Additionally, longitudinal studies are essential to assess the long-term impact of GABA on the progression of PCOS and associated health risks. Mechanistic studies should further explore the interaction between GABA and other neuroendocrine pathways. Standardizing diagnostic criteria, employing validated outcome measures, and utilizing rigorous study designs, with adequate sample sizes and control groups, will be crucial for all future research endeavors.