Cannabis and Endometriosis: The roles of the gut microbiota and the
endocannabinoid system

1.1. Endometriosis and the Endocannabinoid System

Figure 1. The endocannabinoid system (ECS) in pain. As the ECS is spread throughout the central and peripheral nervous system, a psychological effect is reported as a result of endocannabinoid dysregulation. This is coupled with the menstrual-cycle dependent hormonal variation occurring due to varying levels of cannabinoid receptor 1 (CB₁) and cannabinoid receptor 2 (CB₂). This figure is attributed to [36].

Figure 1. The endocannabinoid system (ECS) in pain. As the ECS is spread throughout the central and peripheral nervous system, a psychological effect is reported as a result of endocannabinoid dysregulation. This is coupled with the menstrual-cycle dependent hormonal variation occurring due to varying levels of cannabinoid receptor 1 (CB₁) and cannabinoid receptor 2 (CB₂). This figure is attributed to [36].

Research into the Cannabis genus during the 1990s contributed to the scientific discovery of ECS [37]. The ECS is a complex signaling system that is comprised of three major components: G-protein-coupled cannabinoid receptors (CB₁, CB₂, endocannabinoids (endogenously produced cannabinoids, including anandamide (AEA) and 2-arachidonoyl glycerol (2-AG)), ion channel transient receptor potential vanilloid 1 (TRPV1)[38] and the enzymes involved in the synthesis and catabolism of endocannabinoids [39,40,41].

Research to date has demonstrated that the ECS is involved in homeostasis and regulation via neuromodulatory activity, physiological processes such as digestion [42], immune function [43], nociception [44], appetite regulation[45], cardiovascular and respiratory function[46] and sleep wake cycles[47].

The ECS influences pain modulation, making it a potential target for the treatment of chronic pain conditions such as endometriosis (Figure 1). Activation of CB₁ and CB₂ receptors by endocannabinoids or exogenous cannabinoids can suppress nociceptive processing and induce analgesia[48,49,50,51]. Similar observations were made in preclinical studies showing the analgesic effects of cannabinoid agonists on neuropathic pain[52,53,54]. However, clinical trials have presented mixed results in terms of the efficacy of cannabinoids in pain modulation. In a prospective randomized placebo-controlled trial, smoked cannabis (3.56% THC – participants smoked three cigarettes daily over a 4-day period) reduced daily pain experienced by adults with HIV-associated sensory neuropathy[55]. In contrast, the administration of Sativex, a low-dose THC (2.7mg/100μL) and CBD (2.5mg/100μL) combination oro-mucosal spray (used over an 8-week study period – drugs were titrated per patient) in adults with multiple sclerosis showed non-significant differences between the treatment and placebo group[56]. As studies often employ different modes of administration, which affects bioavailability, the resulting effect of cannabis varies, therefore further studies will be required to fully understand the mechanisms and therapeutic potential of the ECS.

Endocannabinoid receptors have been found throughout female reproductive tissue[41] and the use of exogenous cannabinoids may alleviate endometriosis-associated pain [57,58]. The ECS also regulates endometriosis mechanisms such as inflammation, angiogenesis, apoptosis, endometrial hyperproliferation and fibrosis [41,57,58,59,60,61,62,63], and this may be a potential pathway by which endometriosis lesions re-occur post surgery.

Cannabinoid receptors, specifically CB₁ receptors, are expressed on both the somata and fibres of sensory and sympathetic neurons that innervate endometriotic growths[64]. Activation of CB₁ receptors has been linked to a reduction in endometriosis-induced hyperalgesia, while blocking CB₁ receptors increases pain[65]. Moreover, elevated levels of endocannabinoids, such as AEA and 2-AG, have been observed in people with endometriosis[41]. However, the expression of CB₁ receptors in endometrial stromal cells is lower in endometriosis compared to healthy controls, suggesting a negative feedback loop that may impair the ability of endocannabinoids to control pain[66]. The levels of endocannabinoids in people with endometriosis vary across the menstrual cycle, and this is thought to be influenced by the on-demand synthesis and degradation of enzymes[67]. In the secretory phase, however, CB₁ regulation is absent, reflecting an impaired response to progesterone levels[68]. Table 1 outlines the findings of these key studies.

1.1.1. Endocannabinoid Deficiency in Endometriosis

Endometriosis has been described by some researchers as an “endocannabinoid deficiency”[68]. Plasma levels of endocannabinoid ligands AEA, 2-AG, N-oleoylethanolamine (OEA) and N-palmitoylethanolamine (PEA) fluctuate in people with endometriosis[68]. When associating these endocannabinoids with endometriosis-associated pain severity, a correlation was noted between both increased endocannabinoid ligands in plasma and decreased local CB₁ receptor expression in people with endometriosis.[68]. These levels were studied in association with common endometriosis symptoms, where it was identified that elevated levels of AEA was linked with moderate-to-severe dysmenorrhea while elevated levels of PEA were found in people with moderate-to-severe dyspareunia[68]. This indicates a potential dysregulation in the ECS, suggesting an altered signalling process in response to endometriosis-related pain.

1.1.2. Potential Interplay of the ECS and Endometriosis

The peritoneal microenvironment is often studied in light of the establishment and progression of endometriosis. Inflammatory cytokines (interleukin-1β (IL-1β), interleukin-6 (IL-6), insulin-like growth factor (IGF-1) and tumour necrosis factor-α (TNF-α) are observed to be at a higher concentration in the peritoneal fluid of people with endometriosis [40,69,70,71]. The production of proinflammatory cytokines may occur as a result of disease-modified macrophages producing IGF-1, as high concentrations of IGF-1 were found in the peritoneal fluid of people with endometriosis[15]. This in turn leads to people experiencing hyperalgesia, which has been correlated with altered expression of TRPV1 in the peritoneum of people with endometriosis [72]. Levels of 2-AG and AEA have also been observed to be significantly higher in the peritoneal fluid of people with endometriosis, and this was associated with relatively higher abdominal pain[73]. The protective role of the ECS was observed in people with endometriosis, where immunohistochemistry revealed that cannabinoid agonists inhibit endometrial cell proliferation[61]. In contrast, previous studies have also shown the involvement of the ECS in endometriosis modulation and progression. ECS modulation in innervation of ectopic uterine growths was demonstrated through the abundance of CB₁ receptors on sensory and sympathetic fibres innervating ectopic growths, dorsal root ganglia and coeliac ganglia[41]. This was also observed in the epithelial cells in ovarian endometriotic lesions[74]. Such studies demonstrate the complex and contradictory role of the ECS in pain modulation, warranting more research in this area to understand the mechanisms behind the interaction of the ECS and endometriosis.

1.1.3. Exogenous cannabinoids

The intricate and multifaceted role of the ECS in pain modulation has spurred investigations into the potential impact of exogenous cannabinoids for pain management. While clinical trials are yet to be conducted, preclinical studies have explored the effects of the exogenous cannabinoid delta-9-tetrahydrocannabinol (THC) on pain management. In murine models of endometriosis, repetitive administration of botanically-derived THC demonstrated protective properties[75]. It hindered the growth of ectopic endometrial tissue while alleviating mechanical hypersensitivity in the caudal abdominal region. In an experimental mouse model varying concentrations of cannabidiol (CBD) were found to significantly reduce endometriotic implant surface area, alongside proinflammatory cytokine levels, including IL-6 and TNF-α[76]. A similar study showed that CBD administration reduced endometriotic lesion diameter, volume and area in vivo, demonstrating antioxidant effects by reducing lipid peroxidation[77]. These promising findings highlight the potential therapeutic benefits of CBD and THC for endometriosis-associated pain, warranting the need for human studies.

1.2. The Endocannabinoid System and the Gut Microbiota

The gut microbiota refers to the collection of trillions of microorganisms, including bacteria, archaea, viruses and fungi. The gut microbiota plays a crucial role in human health and disease, impacting immunomodulation and inflammatory processes[78].

In humans, the gut microbiota is dominated by four bacterial phyla: Firmicutes, Bacteriodetes, Actinobacteria and Proteobacteria[79]. As the phylotypic composition of these can vary amongst individuals[80], it can be implied that each host has a unique biological relationship with its gut microbiota, thereby influencing the risk of disease [81,82]. The composition of the gut microbiota in physiological processes also changes with age, implicating long-term health outcomes.

Given the nascent nature of this field of research, there is currently a scarcity of studies examining the relationship between the ECS, the gut microbiota and endometriosis. However, studies have shown the involvement of the ECS in the gut microbiota in metabolic and inflammatory disorders.

The gut is part of the ECS. CB₁ receptors have been localised in the gut epithelium, smooth muscle, submucosal myenteric plexus and myenteric ganglia[83,84]. CB₂ receptors have been detected in the plasma cells and macrophages of the gastrointestinal (GI) mucosa and submucosa, as well as intestinal epithelial cells in the GI mucosa[83,85]. While CB₁ receptor activation is associated with appetite regulation and relief from nausea and vomiting, CB₂ receptor activation mediates inflammation[86]. Endocannabinoids are known to be synthesised in various parts of the gut and their levels fluctuate, based on metabolic and inflammatory status. Endocannabinoids and exogenous cannabinoids exert opposite effects on gut permeability. For instance, when examining decreased permeability as a result of inflammation, it was demonstrated that 2-AG and AEA increased permeability, while THC and CBD decreased permeability[87,88]. Recent literature on the link between the ECS and gut microbiota is summarised in Table 2.

1.2.1. Interactions between gut microbial communities and endocannabinoids

While the interactions between gut microbiota and endocannabinoids has mainly been investigated in preclinical models, studies have linked these interactions to beneficial effects in disease states such as inflammatory bowel disorder (IBD). The administration of Lactobacillus acidophilus in mice led to an increase of CB₂ expression in intestinal epithelial cells, resulting in analgesic effects, thus decreasing visceral pain[85]. Furthermore, the administration of a probiotic mixture containing Bifidobacteria, Lactobacilli spp. and Streptococcus thermophilus in zebrafish resulted in an upregulation of CB₁ and CB₂ expression, which then led to anti-inflammatory effects[89,90].

Akkermansia muciniphila is a Gram-negative anaerobic mucus-degrading bacterium, abundantly found in healthy intestinal mucosa. A. muciniphila has been found to modulate gut barrier integrity. Its protective effects have been cited in relation to the ECS. A study administering Akkermansia muciniphila in mice fed a high-fat diet led to an increase in 2-AG, 2-OG and 2-PG levels[91]. A. muciniphila has been shown in regulate CB₁ mRNA in Caco-2 cells through increased production in outer membrane vesicles, which prevent the development of metabolic disorders such as obesity[92]. Moreover, a decrease in CB₁ activity resulted in reduced circulating lipopolysaccharide (LPS) levels, thus improving the inflammatory cytokine profile and intestinal permeability. This occurred alongside enhanced A. muciniphila and Lachnospiraceae levels in the gut. The gut microbiota profile of obesity is characterised by a Firmicutes:Bacteriodetes ratio, whereby there is an increased abundance of Firmicutes and a reduced abundance of Bacteriodetes. When chronically treating obesogenic mice with THC, this ratio was shifted and an increase in the abundance of A. muciniphila was observed, demonstrating the protective effects of exogenous cannabinoids [93].Further investigation is required to understand how A. muciniphila interacts with endocannabinoids and the outcomes of this on inflammation-associated disorders.

Studies have also examined the relationship between the ECS and gut microbial metabolites. For instance, endocannabinoids have been found to mediate the anti-inflammatory effects of SCFAs. This association was observed in an exercise intervention[94] where increase in SCFAs (including butyrate) and SCFA-producing bacteria (such as Bifidobacterium) were correlated with decrease in proinflammatory cytokines TNF-αand IL-6. Furthermore, an increased abundance of SCFA-producing bacteria, such as Bifidobacterium, was positively associated with increases in endocannabinoids AEA, PEA and OEA, which were correlated with CB₁ receptor levels. These studies provided an indication of how the association between ECS and the gut microbiota can be potentially utilised to improve gut inflammation.

1.2.2. The ECS-gut-brain axis

As CB₁ receptors are found in the CNS, the ECS is involved in signalling, modulating various physiologic and homeostatic processes. While the ECS-gut-brain axis has not been studied extensively, recent studies have considered the involvement of the ECS-gut-brain axis in exercise interventions, processes of food intake and disease states such as Alzheimer’s disease.

While investigating the microbiota-dependent gut-brain pathway on exercise, it was shown that the CB₁-expressing TRPV1 sensory neurons are triggered by fatty acid amide metabolites from gut bacteria such as Lachnospiraceae and Eubacterium [95]. The protective effects of Lachnospiraceae have been well-studied in various diseases, including ulcerative colitis[96,97]. This led to analgesic effects observed after exercise, demonstrating the interaction of intestinal microbial colonization and peripheral CB₁ signaling.

Endocannabinoids such as AEA and CB₂ receptors, and enzymes such as fatty acid amide hydrolase (FAAH) influence retrograde signalling (observed in Alzheimer’s Disease) in the brain. Here, inhibitory feedback regulates neurotransmitter release[98]. Moreover, as AEA induces gut permeability, its overexpression is thought to cause a ‘leaky’ gut, resulting in metabolic endotoxemia[99]. As this condition leads to the release of toxins such as LPS which may cross the epithelial barrier, an inflammatory signalling pathway may develop, impacting the CNS and resulting in neuroinflammation[100].

CB₁ binding leads to increased food intake. CB₁ receptors are present in cells of the lining of the intestinal epithelium. When CB₁ receptor activity is heightened, in the small-intestinal epithelium, the release of cholecystokinin-8 (CCK-8) is inhibited, resulting in delayed satiation and overeating (in diet-induced obesity)[101], highlighting the indirect control mechanism of endocannabinoids in gut-brain neurotransmission.

1.2.3. The ECS in IBD

The use of cannabis for abdominal pain relief and other IBD symptoms is prevalent, with studies reporting use amongst 70-95% of people with IBD [86,102,103,104]. However, several studies have reported similar results when using endocannabinoids and/or exogenous cannabinoids in therapies for IBD. In analysis of biopsies of paediatric patients, AEA levels were found to be significantly decreased in inflamed IBD mucosa[105]. A significant increase in the expression of CB₂ receptors has also been observed in the biopsies of Crohn’s disease ileum and in rectum biopsies of ulcerative colitis, colocalised with T-lymphocyte infiltration [106] (Figure 2). Similarly, a murine IBD studies demonstrated that increasing the availability of the endogenous CB₁ and CB₂ receptor agonists, diminishes visceral pain[107,108,109]. Nonetheless, the issue remains that such implications have not translated to clinical trials, as noted in one study where people with IBD using cannabis reported higher incidences of abdominal pain and arthralgias[110]. Further research is required to develop insights into what may be causing these negative side effects and utilisation of the ECS as a therapy for IBD.

There is a high prevalence of inflammatory bowel syndrome (IBS) and its associated symptoms in people with endometriosis, in one study 52% (194/373) of those with endometriosis had diagnosed IBS[111], where those experiencing minimal-mild endometriosis often report more severe IBS symptoms, as compared to those with moderate-severe endometriosis. Similarly, of 160 people with diagnosed IBS, 59 had a history or recent diagnosis of endometriosis[112]. People with endometriosis are reported to have a threefold increase in likelihood of developing IBS[113]. Such studies demonstrate the importance of understanding the mechanisms through which inflammatory bowel diseases and endometriosis interact and thus the need for treatments targeting both diseases.

A bidirectional relationship between endometriosis and gut microbiota has been proposed (Figure 3). Current literature on the potential role of gut microbiota in endometriosis is summarised in Table 3. As gut microbes and their metabolites are involved in various immune, metabolic and epithelial functions, imbalances in the gut microbiota can trigger an inflammatory response through specific inflammatory immune cell recruitment, proinflammatory cytokine production and compromised immune surveillance. This has been observed in different disease states such as IBD [114], psoriasis[115] and cancer[116].

1.3.1. Microbial dysbiosis in endometriosis

In the endometriotic state, larger percentages of bacterial phyla Proteobacteria, Verrucomicrobia, Streptococcus or Fusobacteria have been reported. Increases in Enterobacteriaceae, Streptococcus and E. coli have been identified as dominating phyla in endometriosis cohorts[117]. Recently, Fusobacterium is suggested to contribute to the pathogenesis of endometriosis[118]. The dominance of Shigella has also been noted[119]. Such microbial communities are known to be involved in the degradation of estrogen by producing β-glucuronidase and β-glucosidase[120], resulting in the development of a high estrogen environment, promoting the progression of endometriosis[121,122].

An increase in E. coli is attributed as a biomarker of endometriosis and this has led to the “bacterial contamination” theory. High levels of E. coli have been found in faecal[123] and menstrual blood samples[124] of participants with endometriosis. E. coli is known to trigger TLR4 mediated growth and progression of endometriosis, resulting in pelvic inflammation[125]. This occurs through the production of LPS, triggering secretion of secondary inflammatory mediators such as NF-κB, in the peritoneal cavity, therefore, resulting in the development and progression of endometriosis.

Further research is required to unravel the intricate dynamics between the microbiota and its influence on endometriosis progression. Such investigations would not only grow the understanding of the way in which the microbiota promote endometriosis pathogenesis, but also the development of targeted therapies to improve the quality of life of those with endometriosis.

1.3.2. Microbial dysbiosis and endometriosis symptoms

Research has demonstrated correlations with specific microbes and bacterial phyla impacting symptoms in those with endometriosis. Previous studies have shown the estrogen-gut-brain axis, which is thought to influence the development of chronic stress in people with endometriosis via the activation of β-adrenergic signalling[126]. This has been associated with dysbiosis on a genus level, particularly decrease in Paraprevotella, Odoribacter, Veillonella and Ruminococcus [126] which are often viewed as biomarkers of endometriosis[127]. A murine study showed that a decrease in Ruminococcus is negatively correlated with apoptosis of endometriotic epithelial cells and increased IL-6 levels, resulting in peritoneal inflammation[128]. Prevotella has been found in high abundance in people with endometriosis, especially in those experiencing gastrointestinal symptoms, and is associated with constipation, bloating, flatulence, vomiting and nausea[129]. As many studies report a correlation between gut microbiota imbalances and endometriosis, continued and extensive research is essential in understanding the full potential of harnessing the gut microbiota for the treatment of endometriosis and its associated symptoms.

The vaginal microbiota is dominated by healthy Lactobacillus, which maintains an acidic and protective environment, preventing the growth of pathogenic bacteria. However, in endometriosis, the protective environment maintained by vaginal microbiota is impacted by an abundance of Gardnerella, Escherichia, Shigella, Ureoplasma[119], Streptococcus, Moraxellae, Staphilococcus and Enterobacteria, coupled with a lowered abundance of Lactobacillus[130]. These imbalances have been associated with endometriosis-associated pelvic pain[131,132]. An understanding of the vaginal microbiota in endometriosis, in correlation with the ECS, is required for the characterisation of treatments that may be prescribed for people with endometriosis.

6. Conclusions and Future Directions

This review summarises the relationship between endometriosis, the ECS and the gut microbiota. While studies have not completely deciphered the molecular basis of these relationships, current literature demonstrates the vital roles that the ECS and the gut microbiota play in potential implications in preventing the development of endometriosis as well as effective therapeutic strategies. The mechanisms of the ECS in both endometriosis and the gut microbiota were explored. Further research understanding the mechanisms, influence on inflammation and analysis of endocannabinoids and exogenous cannabinoids is required for the development of treatments of various diseases including endometriosis. While the impact of cannabinoids in endometriosis has been investigated, future studies are needed to comprehend how cannabinoids influence endometriosis-related pain and symptoms and how this can be implemented in the clinic, following efficacy and safety studies. This may be done through implementation of exogenous cannabinoids, which have been shown to have protective effects. An understanding of various inflammatory diseases was used to show the potential therapeutic role of the gut microbiota, while paving new avenues for further research. Finally, the relationship between the gut microbiota and endometriosis was explored, highlighting the need for further investigations of how cannabinoids may influence gut microbiota in endometriosis.