Recurrent pregnancy loss is associated to leaky gut: a novel pathogenic model of endometrium inflammation?

Spontaneous pregnancy loss is a surprisingly common occurrence. Approximately 15–20% of all clinically recognized pregnancies result in spontaneous loss and it is believed that there are many more pregnancies, up to 40–50%, that fail prior to being clinically recognized [1]. For a single isolated miscarriage within the 10th week of gestation no tests should be proposed to couples. A completely different clinical condition is represented by recurrent pregnancy loss (RPL), which occurs in 3–5% of all women and is defined by three consecutive pregnancy losses prior to 23 weeks of gestational age [1].

Using evidence-based tests for evaluation of couples with RPL, approximately 60% of couples will have an etiology identified that could be associated with their losses. There are other factors, such as lifestyle factors, that may contribute to miscarriage and can be corrected or eliminated. These include maternal age, obesity, tobacco use and alcohol consumption. However, in about 40% of all RPL cases, after a complete screening for the known causes or predisposing factors, none of the above anomalies can be found, identifying cases of idiopathic RPL [1]. Then, despite a thorough evaluation for RPL, not all couples will have answers to their dilemma.

It is known that RPL has a significant emotional impact on couples: early pregnancy loss is usually a shocking and traumatic event for women and their families. During the initial weeks following a loss, symptoms of grief may be impossible to distinguish from depression, and some women may continue to experience depressive symptoms for months [2]. Contributing to the distress experienced after miscarriage is the fact that society may not recognize the significance of the loss to the parents. Recently, Kolte and coauthors found that both psychological stress and major depression are significantly more common among women with RPL than in those with regular pregnancies [3]. From a pathogenic point of view, it is worth of note that there are evidences showing that depression and inflammation are intertwined and feeding off each other. This bidirectional loop, in which depression facilitates inflammatory responses and inflammation promotes depression may have consequences on health. Indeed, elevated inflammatory signaling disregulates neurotransmitter metabolism, impairs neuronal health, and alters neural activity in mood-relevant brain regions [4, 5]. In particular, it has been shown that pro-inflammatory cytokines and lipopolysaccharide (LPS) may induce depressive symptoms [6‐8]. On the other hand, depression is accompanied by an activation of the inflammatory response system. There is now evidence that intestinal mucosal dysfunction, characterized by an increased translocation of Gram-negative bacteria, called “leaky gut”, plays a role in the inflammatory pathophysiology of depression [9]. Furthermore, it is suggested that the increased LPS translocation, related to leaky gut, may induce an immune response and a systemic tissue inflammation in patients with depression and specific sickness behaviour symptoms [10].

Concerning pregnancy loss, the endometrial expression of pro-inflammatory cytokines (IL-1β, TNF-α, IFN-γ, and TGF-β1) has been shown to be up-regulated in women with idiopathic RPL compared with controls [11]. Moreover, recently, we observed an abnormal activation of the endometrial inflammasome Nalp-3, which represents the first line of defense against cellular stress and is a crucial component of innate immunity [12]. Upon activation of Nalp-3, apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1 are assembled and this multiprotein complex enables the caspase-1-mediated proteolytic processing of the pro-inflammatory cytokines (IL-1β and IL-18), generating their respective mature secretory forms. These events are necessary for the induction of further systemic responses and spreading of inflammation [13, 14].

Currently, there are no available data in the international literature on the role of abnormal intestinal permeability and RPL. Thus, since the clinical conditions characterized by increased intestinal permeability, also called “leaky gut”, may be related to the passage of pathogens-derived proteins (like LPS) into the blood circulation, therefore inducing inflammasome activation, the aims of this study were:

⁵¹Cr-ethylene-diamine-tetraacetic acid (⁵¹Cr-EDTA) absorption test was performed in all patients enrolled to assess intestinal permeability as described elsewhere [19]. Enrolled women were all advised to avoid pregnancy during the test month. After an overnight fast, patients were administered 0.37 MBq ⁵¹Cr-EDTA (Amersham Health, England, UK) in 10 ml water. The radiation dose administered was very low, analogue to the dose coming from a 2 days exposure to natural background radiation. The 24-h urinary excretion of ⁵¹Cr-EDTA was analyzed using a γ-counter (LKB-Wallac 1282 Compugamma, Turku, Finland). Cr-EDTA clearance was calculated using the formula: Results were expressed as fraction of the oral administered dose and considered abnormal when ≥ 3% [19]. All women with abnormal intestinal permeability test (≥ 3%) underwent a gastroenterological evaluation and were then evaluated for serum levels of immunoglobulin (Ig) A and E, anti-endomysial, anti-gliadin, anti-transglutaminase, perinuclear and cytoplasmic anti-neutrophil cytoplasmic antibodies (pANCA, and cANCA, respectively), Anti-Smooth Muscle and Anti–Saccharomyces cerevisiae antibodies (ASMA and ASCA, respectively) antibodies, HLA DQ2-DQ8 haplotype and underwent lactose/lactulose/sorbitol breath test and fecal calprotectin assay to exclude all known causes of increased intestinal permeability.

The main finding of the present study is the higher prevalence of abnormal intestinal permeability in patients with idiopathic RPL. In the same population, we detected higher plasma levels of LPS, an immunogenic parietal fragment from Gram-negative bacteria. Furthermore, consistently with a previous study [12], we confirmed an increased expression and activation of Nalp-3 inflammasome proteins in the human endometrium of women with RPL, as well as an increased caspase-1 activation and secretion of the pro-inflammatory cytokine IL-1β. Interestingly, for the first time, we could link the endometrial Nalp-3 inflammasome over expression and activation with leaky gut. Based on our data, we can speculate that in RPL women bacterial components might enter systemic circulation through a damaged intestinal barrier and induce inflammatory cytokines production in endometrial tissues, via inflammasome Nalp-3 activation. The lack of significant correlation between increased intestinal permeability and LPS might be explained: (a) because of the small number of patients recruited; (b) for the heterogeneity of gut microbiota composition in our population; (c) because not only bacteria-derived molecules but also other molecules coming from the external environment (food, chemicals, drugs, etc.) might cross gut barrier and stimulate Nalp-3 inflammasome. Thus, we hypothesize that leaky gut, occurring for reasons that we cannot explain yet, allows the passage of antigens through the intestinal barrier, that might elicit innate immunity in endometrial tissue. Hence, when there is an increased intestinal permeability and/or when higher circulating levels of LPS are detected in RPL subjects, there might be a higher risk of endometrial inflammation and reproductive disorders.

The occurrence of cross-talk between the gut and the reproductive system may be an intriguing hypothesis that could suggest a possible new approach to a specific group of patients with idiopathic RPL. Evidence supporting a role for the intestinal-endometrial axis in the pathogenesis of early pregnancy complications has being slowly accumulating over the most recent years [20, 21]. The most studied pathogenic model is celiac disease (CD). CD has been related to augmented intestinal permeability due to the ability of gliadin to disrupt intestinal epithelial tight junction proteins including zonulin-1, claudin-1 and occludin, all polyamines with a pivotal role in the control of intestinal barrier function, inducing the leaky gut syndrome [22, 23]. Several studies reported an increased risk of reproductive failures including recurrent miscarriage in women with CD. In particular, according to a previous meta-analysis, women experiencing RPL have an Odds Ratio (OR) for CD of 5.82 (95% CI 2.30–14.74) and the risk of miscarriage in women with CD is significantly higher with an RR of 1.39 (95% CI 1.15–1.67) [20]. As a whole, we can hypothesize that recovering intestinal barrier might reduce tissue inflammation and take the endometrium back to a favorable environment for implantation and pregnancy development.

Finally, the role of anxiety and depression in RPL patients has yet to be clarified. We did not find a strong correlation among depression/anxiety state and intestinal permeability or abnormal LPS levels but only an association between abnormal LPS circulating levels and anxiety trait. However, in our population of RPL, we found a higher prevalence of self-reported stress and moderate/severe depression. Previous studies on depression and RPL have been few, using a variety of scales and lacking an appropriate comparison group [24‐27]. The reported prevalence of depression varies considerably across studies, ranging from 15 to 33% [24‐27]. Psychological stress and RPL have been not extensively studied, but one case–control study described a significantly higher psychological stress total score among 45 patients with RPL, compared with 40 controls [10]. Interestingly, it has been demonstrated that the gut-brain axis involves bidirectional communication between the central nervous system and the gastrointestinal tract via neurocrine and endocrine signaling pathways [28]. Indeed, physical and psychological stressors can alter the gut microbiota’s composition and metabolic activities, and signals produced by the gut microbiota can in turn affect the brain and emotional responses [29]. Moreover, depression can promote intestinal permeability leading to leaky gut. Interestingly, depressed patients have been found to have higher antibodies against gut bacteria than healthy subjects [30]. In another study, patients with major depression showed higher circulating levels of 16S rDNA, a marker of bacterial translocation, compared with healthy subjects, and the magnitude was correlated with depressive symptom severity [31]. As a whole, it is widely accepted that increased bacterial translocation could play a role in the inflammatory pathophysiology of depression by inducing acute inflammatory responses and contributing to subchronic inflammation [32]. To support these evidences, LPS has been shown to elicit depressive and anxious behaviors in rats [33].

Leaky gut, depressive symptoms and RPL are intertwined conditions but it is difficult to establish which cause-effect relationship might occur. This is the first study detecting abnormal intestinal permeability in women with RPL. According to our hypothesis, in this population an abnormal intestinal permeability, worsened or caused ex novo by a depressive or anxious state, might allow passage of immune triggers from external environment, not only of bacterial derivation, that might elicit innate immune response, leading to endometrial inflammation and, thus, potentially to miscarriage.

More studies are needed to confirm our hypothesis and define the pathogenic mechanisms of inflammation-induced miscarriage. Furthermore, the beneficial effect of recovering intestinal barrier function in RPL women, as well as psychological health, on subsequent obstetric outcomes should be evaluated.