Beyond the reproductive tract: gut microbiome and its influence on gynecological health

43 Purpose of review: The analysis of microbiome in association with female health is today 44 a “hot topic” with the main focus on microbes in the female reproductive tract. 45 Nevertheless, recent studies are providing novel information of the possible influence of 46 the gut microbiome on gynecological health outcomes, especially as we start to 47 understand that the gut microbiome is an extended endocrine organ influencing female 48 hormonal levels. This review summarizes the current knowledge of the gut microbes in 49 association with gynecological health. 50 Recent findings: The gut microbiome has been associated with endometriosis, 51 polycystic ovary syndrome, gynecological cancers, and infertility, although there is a lack 52 of consistency and consensus among studies due to different study designs and protocols 53 used, and the studies in general are underpowered. 54 Summary: The interconnection between the gut microbiome and reproductive health is 55 complex and further research is warranted. The current knowledge in the field emphasizes 56 the link between the microbiome and gynecological health outcomes, with high potential 57 for novel diagnostic and treatment tools via modulation of the microenvironment. 58 59 Keywords: microbiota, endometriosis, polycystic ovary syndrome, cancer, infertility 60 61 62 63 64 4 1. INTRODUCTION 65 The human gastrointestinal tract harbors trillions of microorganisms including bacteria, 66 archaea, viruses, protozoa, and fungi, being the largest and most diverse microbial 67 ecosystem within the human body. The collection of the intestinal microbial genomes, 68 the gut microbiome, is a research field of increasing interest since it represents a genetic 69 pool more than one order of magnitude higher in genes than the human genome [1]. The 70 vast majority of these microbial communities co-evolved symbiotically with the host and 71 contribute to important metabolic, immune and epithelial functions, being crucial for the 72 host physiology and pathophysiology [2–4]. 73 Recent evidence refers to the gut microbiome as an extended endocrine organ due to its 74 profound interaction with hormone levels [5]. Furthermore, a sex bias has been identified 75 in microbiome-related diseases which are associated with sex hormones [6]. In this 76 context, the term “microgenderome” has emerged to define the interactions between the 77 microbiome, sex hormones and the immune system [7]. This interaction could unravel 78 the molecular mechanisms underlying the gut microbiome influence on female 79 reproductive health and how its dysbiosis could lead to different pathologies [8]. 80 The gut microbiome-estrogen axis has been proposed as a cornerstone implicated in the 81 pathogenesis of different gynecological conditions, such as polycystic ovary syndrome 82 (PCOS), endometriosis, gynecological cancer, infertility and adverse pregnancy 83 conditions [9] (Figure 1). This crosstalk between the gut microbiome and estrogens is 84 regulated by the estrobolome, the aggregate of gut bacterial genes whose products are 85 capable of metabolizing estrogens [10]. Certain enteric bacteria secrete β-glucuronidase, 86 the main estrogen-regulator of the estrobolome, that converts the conjugated estrogen 87 (glucuronic acid) into its deconjugated form that exerts its biological activity [11]. Thus, 88 5 an optimal β-glucuronidase activity reduces the inactivation of estrogen, leading to a 89 balanced hormone circulating levels. However, a reduction in the gut microbial diversity 90 as a result of dysbiosis and inflammation could reduce the β-glucuronidase activity and 91 this reduction has been linked to hypoestrogenic pathologies such as obesity, metabolic 92 syndrome, cardiovascular disease and cognitive decline [9]. Otherwise, an increased β-93 glucuronidase activity is associated with hyperestrogenic conditions and can lead to the 94 progression of gynecological estrogen-driven diseases [9] (Figure 1). Since the 95 estrobolome may have a profound influence on these pathologies, future interventions 96 targeting the estrobolome-microbiome axis emerge as promising diagnostic and treatment 97 tools for women’s health [5]. However, the molecular mechanisms underlying the 98 relationships between the gut microbiome, estrogen metabolism and gynecological 99 outcomes are still in its infancy. In this narrative review, we summarize the whole body 100 of knowledge of the gut microbiome and its interactions with different gynecological 101 health conditions. 102 103 2. ENDOMETRIOSIS 104 Endometriosis is defined as an estrogen-dependent chronic inflammatory gynecological 105 disease characterized by endometrial-like tissue present outside of the uterus. It represents 106 a major health concern since it affects 6-10% of women in reproductive age [12]. 107 Regardless of the continuous research, the exact mechanisms of endometriosis are still 108 undetermined. The most widely accepted hypothesis for the origin of endometriosis is 109 Sampson's retrograde menstruation, which explains that women commonly have 110 retrograde menstrual flow [13]. Nevertheless, only 10% of women are diagnosed with 111 endometriosis [14]. Growing evidence proposes a multifactorial origin for endometriosis 112 6 development driven by genetic predisposition, environmental factors, inflammation, 113 immune activation, hormone dysregulation and microbial dysbiosis [15, 16]. 114 Much is known of the role of the gut microbiome in maintaining the integrity of the 115 gastrointestinal epithelial lining and immune balance to prevent bacterial translocation, 116 which can cause low-grade systemic inflammation [17]. While it is well-established that 117 the gut microbes influence immunomodulation and the development of various 118 inflammatory diseases [18], current studies have highlighted the potential implication of 119 enteric microbes in the pathogenesis of endometriosis [11, 19]. The “bacterial 120 contamination hypothesis” proposes that besides estrogen regulation, the gut microbiome 121 could contribute to the onset of endometriosis through lipopolysaccharide (LPS) 122 endotoxin as the initial trigger and bacterial contamination as its source in the intrauterine 123 environment [20]. LPS is found in the cell wall of Gram-negative bacteria, and is a marker 124 of inflammation which has been linked to endometriosis lesions activating the immune 125 response by binding with Toll-like receptor 4 [20]. A systematic review concluded that 126 increased abundance of Proteobacteria, Enterobacteriaceae, Streptococcus and 127 Escherichia in the gut associated with the presence of endometriosis [21] (Figure 1). In 128 line, a Shigella/Escherichia dominant gut microbiome in women with advanced stages 129 3/4 endometriosis have been described [22], and an increase in Streptococcus in the gut 130 of patients with the 3/4 endometriosis stages has been reported [23]. Further, a recent 131 study detected a higher abundance of Shigella flexneri (Proteobacteria phylum) in 132 patients with external genital endometriosis compared to controls [24]. Additionally, a 133 higher proportion of Gram-negative bacteria belonging to Desulfobacterota phylum have 134 been detected in women with endometriosis when compared to healthy controls [25]. 135 Proteobacteria and Desulfobacterota phyla are both characterized by Gram-negative 136 staining, and, therefore, presenting LPS in the outer membrane [26]. Interestingly, a 137 7 recent translational study demonstrated a pathogenic mechanism via Fusobacterium (a 138 Gram-negative bacterial genus) infection in endometrial cells through activation of 139 transforming growth factor–β (TGF-β) signaling [27]. This activation leads to the 140 transition from quiescent fibroblasts to transgelin (TAGLN)–positive myofibroblasts, 141 which are able to proliferate, adhere, and migrate in vitro [27]. It was also observed that 142 inoculation of Fusobacterium nucleatum in a murine model of endometriosis resulted in 143 increased numbers and weights of endometriotic lesions [27]. 144 Short-chain fatty acids (SCFAs) are microbial metabolites with pleiotropic beneficial 145 effects for the host metabolism and immune regulation through their action on T-146 regulatory cells [28, 29]. Lachnospiraceae, Eubacteriaceae and Ruminocacceae family 147 members are the main producers of SCFAs in the intestine, particularly producing acetate 148 and butyrate [30, 31] (Figure 1). Several studies have found lower abundances of 149 butyrate-producing microbes such as Lachnospira, Ruminococcus, Eubacterium eligens 150 and Coprococcus catus in women with endometriosis [23, 24, 32, 33]. In particular, 151 butyrate has been described as an anti-inflammatory mediator that could indirectly 152 regulate endometriosis-related symptoms such as visceral inflammatory pain [34]. 153 Considering the connection between the dysbiosis (through an increase of Gram-negative 154 bacteria and/or depletion of SCFAs producers) and immune dysfunction, future studies 155 are needed to study whether imbalances within these gut microbes are the cause, 156 consequence or enhancer of endometriosis. 157 Given the hyperestrogenic conditions associated with endometriosis, there is growing 158 interest focused on the estrobolome as a key factor contributing to the progression of the 159 disease. Alterations in the gut microbiome that result in overexpression of estrobolome 160 associated genes could both trigger formation and maintenance of the lesions. 161 Interestingly, β-glucuronidase activity has been found in Gram-negative bacteria [25]. 162 8 Moreover, an analysis of microbial genomes associated enteric Bacteroides, 163 Bifidobacterium, Escherichia and Lactobacillus with β-glucuronidase production [35] 164 (Figure 1). In endometriosis population, several studies have reported higher abundances 165 in these bacteria [23, 32]. Nevertheless, an enzymatic activity study of fecal samples did 166 not reveal significant differences in β-glucuronidase activity in women with and without 167 endometriosis [36]. We have recently performed the first whole metagenome study on a 168 cohort of 1,000 women with and without endometriosis, and did not find any microbial 169 features (species or pathways) associated with the disease [37, 38]. Furthermore, we 170 analyzed the estrobolome-associated genes and did not find any significant differences 171 between the two study groups [38]. 172 Altogether, the previous results pave the way for future strategies targeted to the gut 173 microbiome-estrobolome axis, nevertheless the studies in the field lack consensus and the 174 identification of an endometriosis-associated microbiome profile still constitutes a debate 175 without a definitive answer. It is clear that endometriosis is a complex heterogenic disease 176 and further studies applying well defined, adequately powered study groups are warranted 177 to determine the core microbial composition in endometriosis. 178 179 3. POLYCYSTIC OVARY SYNDROME 180 The polycystic ovary syndrome (PCOS) is one of the most prevalent endocrine disorders 181 in women of reproductive age, affecting up to 20% of women [39]. Despite its high 182 prevalence, its multifactorial complexity has made it challenging to understand the 183 underlying etiology. Possible triggers include genetic factors, intrauterine environment, 184 lifestyle, and, in an increasingly explored approach, alterations in the gut microbiome [40, 185 41]. 186 9 Considering the role of the gut microbes in metabolic disorders, the search of the link 187 between the gut microbes and PCOS is plausible. Numerous studies have detected 188 alterations in the microbial richness, diversity, and microbial composition in PCOS [41]. 189 Specifically, PCOS patients have been observed to exhibit a decrease in α-diversity 190 indices (diversity within a sample) compared to controls [42–45]. Regarding β-diversity 191 (dissimilarity of the microbial community between samples), previous studies have 192 identified differences in the microbial composition among samples from PCOS patients 193 compared to healthy controls [42, 44–47]. While other studies have not observed any 194 significant differences in microbial composition in PCOS [48–52]. Our recent systematic 195 review summarizes that the women PCOS have decreased microbial diversity in the gut 196 and that the prevalent taxa are Bacteroides spp., Parabacteroides spp., Prevotella, 197 Megamonas spp., Megasphaera massiliensis, Escherichia/Shigella, while 198 Bifidobacterium spp., Lactobacillus spp., Faecalibacterium, and Blautia are reduced 199 [41]. 200 The presence of obesity and insulin resistance also emerges as crucial factors in the study 201 of the gut microbes in PCOS. Obesity, a common characteristics in PCOS patients, has 202 been analyzed in relation to the intestinal microbiome, revealing significant differences 203 in the gut microbiome β-diversity in PCOS patients with obesity compared to non-obese 204 patients [53, 54]. In PCOS groups with high BMI, an increased abundance of 205 Erysipelotrichaceae_UCG-003 [45], Prevotellaceae [54], Streptococcus, Fusobacterium, 206 Rhizobacter, and Achromobacter [49] was observed. Nevertheless, other previous studies 207 have not observed any microbiome differences between the groups [48, 49, 51]. 208 Regarding insulin resistance, studies have shown a decrease in the α-diversity when 209 compared to controls [48, 54, 55], and the most abundant bacteria such as Prevotella, 210 10 Megamonas, Dialister [54], Prevotella stercorea [48] and Faecalibacterium [55] have 211 been identified in PCOS patients with insulin resistance. 212 Beyond the bacteria, a number of studies have explored the mycobiome and virome in 213 PCOS patients, where the increased abundance of fungi: Saccharomyces, Lentinula, and 214 Aspergillus [46], Candida, Malassezia, Kazachstania, Microascus, Coniochaeta, 215 Xepicula, Paraphoma, Pyrenochaetopsis, Cephaliophora, Epicoccum, and Sclerophora 216 has been observed in PCOS patients [45]. The analysis of the gut virome detected lower 217 viral diversity and significant alterations in virome composition in women with PCOS, 218 where the most enriched taxon was Quimbyviridae when compared to healthy controls 219 [56]. 220 Altogether, there seems to be a common trend of reduced microbial diversity in PCOS, 221 nevertheless, the studies are performed on limited sample size and lack consensus. 222 223 4. GYNECOLOGICAL CANCER 224 Within the recent years, there has been a growing interest in understanding the connection 225 between the human microbiome and various types of cancers, including gynecological 226 cancers. Among these, the most common are endometrial, cervical, and ovarian cancers, 227 where the endometrial cancer is the most prevalent [57]. However, the most lethal is 228 ovarian cancer, accounting for 5% of total cancer-related deaths [8, 57]. These cancers 229 are characterized by being estrogen-mediated tumors [58]. Estrogens have the capacity to 230 modulate the inflammatory response and increase the production of pro-inflammatory 231 mediators (IL-6 and TNF-α) [59]. This can establish a feedback loop that influences the 232 expression of enzymes associated with ovarian steroidogenesis. The gut microbiome is 233 able to metabolize these estrogens, increasing their concentration and thereby enhancing 234 11 the development of endometrial cancer [59]. Simultaneously, this increase in the estrogen 235 levels also has the potential to induce changes in the gut microbiome, indirectly 236 contributing to the cancer progression [8]. 237 Although there is still limited literature of the relationship between the gut microbiome 238 and endometrial cancer, existing studies provide contradicting results. One study found a 239 significant reduction in the gut microbial α-diversity and differences in β-diversity among 240 endometrial cancer patients [60], while other did not detect any significant differences 241 [61]. These discrepancies are also reflected in the phylogenetic composition, with 242 variations in the abundances of Firmicutes, Proteobacteria, Actinobacteria, and 243 Bacteroidetes in the gut microbiome among endometrial cancer patients when compared 244 to controls [60, 61]. 245 In ovarian cancer studies, changes in β-diversity were consistently observed between 246 patients and controls, however, in richness, no significant differences were detected 247 [62,63]. Moreover, increase in specific bacterial abundances such as Firmicutes, 248 Proteobacteria, and Bacteroidetes phyla have been reported in ovarian cancer patients 249 [62]. 250 The research of the gut microbiome in gynecological cancers is very preliminary and 251 future research is needed to clarify the potential cancer-associated microbial profile and 252 unravel the complexity of the relationship between gut microbes and gynecological 253 cancers. 254 255 5. INFERTILITY 256 The vaginal microbiome has been the subject of extensive research in relation to female 257 fertility; however, the influence of the gut microbiome is still relatively unstudied. Recent 258 12 investigations suggest that the gut microbiome may play a crucial role in the modulation 259 of the reproductive system through the gut-uterus axis. It has been observed that even a 260 small alteration in the commensal and symbiotic gut microbes can trigger dysbiosis, 261 disrupting intestinal homeostasis and increase the risk of inflammatory processes 262 associated with adverse reproductive pathologies [64] (Figure 1). 263 A previous study highlighted that the diversity and composition of the intestinal 264 microbiome, along with its metabolite profiles, show significant alterations in patients 265 who had experienced spontaneous abortions [64]. When analyzing the fecal microbiome 266 in association with spontaneous abortions, an overrepresentation of various opportunistic 267 pathogens (Prevotellaceae_NK3B31_group, Bacteroidales_S24_7_group, and 268 Eubacterium ruminantium) was identified in the affected group, while other 269 microorganisms (Prevotellaceae, Prevotella_1, and Gammaproteobacteria) were more 270 abundant in the control group [64]. Additionally, a significant correlation was found 271 between the metabolites associated with these microorganisms and an increase in 272 cytokines linked to Th1 and Th17 [64]. The reduction in the richness and diversity of the 273 microbiome in patients who had suffered abortions supported these findings, 274 corroborating previous results linking microbiome composition to infertility [65-67]. 275 In a similar context, notable differences in the composition of the gut microbiome 276 between patients with infertility (recurrent implantation failure –RIF- and unexplained 277 infertility) and controls have been detected [65]. Bacteroides and Hungatella stood out as 278 the most abundant genera in the gut in infertilite women, especially in cases of 279 unexplained infertility. A decrease in the genera Prevotella 9, Ruminococcaceae UCG-280 004, Ruminococcaceae UCG-010, and an increase in Bacteroides, Dorea oral clone 281 FR58, and Peptoniphilus were detected in the gut microbiome when compared to controls 282 [65]. Further studies have detected higher abundance of Verrucomicrobia, and members 283 13 of Barnesiellaceae and Phascolarctobacterium in the gut [67], while the genera 284 Stenotrophomonas, Streptococcus, and Roseburia showed a decrease in patients with 285 infertility [67]. Altogether, regardless of these preliminary studies, there seems to be a 286 consensus in the increase of the gut Firmicutes/Bacteroidetes ratio in infertile women 287 when compared to controls [64, 66]. 288 289 6. CONCLUSIONS 290 There is growing body of evidence demonstrating that the gut microbes play important 291 role in female physiology and pathophysiology and that via its endocrine and hormonal 292 regulation, specifically estrobolome regulation can influence female reproductive health. 293 In this review we gather the knowledge of the gut microbiome involvement in 294 endometriosis, PCOS, cancer and infertility, and with the time, the list of different 295 gynecological disorders in association with the gut microbiome will definitely grow. 296 The current knowledge of the microbe-disease associations encompasses the microbial 297 diversity analyses and identification of specific bacterial genera. The majority of the 298 studies of the gut microbiome in female gynecological health have applied the 16S rRNA 299 gene sequencing technique that does not have sufficient specificity to identify bacteria on 300 species level, which makes the generalization of the findings of the current studies 301 imprecise. The whole metagenome sequencing method, although more expensive and 302 requiring advanced bioinformatics skills, would provide more detailed information of the 303 exact bacterial species and detects also other microorganisms within the sample, such as 304 viruses, fungi, archaea and other microeukaryotes. 305 Another important aspect that calls for caution when interpreting the previous findings is 306 that most of the studies have been performed on limited sample size, lacking detection 307 14 power and negative/positive controls. It is known that inherent elements of study design, 308 such as sample size, sample collection method, DNA extraction process, type of 309 sequencing employed, and data analysis, represent limitations that can influence the 310 accurate detection of microorganisms [68, 69]. 311 Furthermore, the currently applied next-generation sequencing-based microbiome 312 analysis techniques assess DNA sequences, which do not necessarily equate with the 313 presence of live bacteria [70]. Thus, DNA-based techniques characterize a microbiome 314 but do not mean that the detected sequences are functionally active microbes. RNA 315 analysis-based technique (i.e. meta-transcriptomics [71] and culturomics [72], together 316 with integration with other omics analysis platforms would provide further knowledge of 317 the functionality of the microbes in the gut in reproductive health and disease. 318 In conclusion, the gut microbiome studies in the female gynecological health are in its 319 infancy, and future research on bigger, well-designed studies together with novel methods 320 are warranted to unravel the core microbial compositions in gynecological health and to 321 understand the function of specific microbes in the disease development. 322 323 ACKNOWLEDGEMENTS 324 This work is part of a Ph.D. thesis conducted in the Biomedicine Doctoral Studies of the 325 University of Granada, Spain. 326 327 FINANCIAL SUPPORT AND SPONSORSHIP 328 This work was supported by the projects Endo-Map PID2021-12728OB-100 and ROSY 329 CNS2022-135999 funded by MCIN/AEI/10.13039/501100011033 and ERFD “A way of 330 15 making Europe”; and by grant FPU19/05561 funded by 331 MCIN/AEI/10.13039/501100011033 and FSE “El FSE invierte en tu futuro”. 332 333 CONFLICTS OF INTEREST 334 The authors have no conflicts of interest. 335 336 16

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Vanstokstraeten R, Demuyser T, Piérard D, Wybo I, Blockeel C, Mackens S. 548 Culturomics in Unraveling the Upper Female Reproductive Tract Microbiota. Semin 549 Reprod Med. 2023. https://doi.org/10.1055/s-0043-1777758. 550 551 552 26 FIGURE LEGEND 553 Figure 1. Potential gut microbiome-driven mechanisms underlying gynecological 554 physiology and pathophysiology. In healthy (eubiotic) conditions, the estrobolome 555 contributes to estrogen activation through the secretion of beta-glucuronidase. 556 Homeostatic circulating estrogen levels regulate menstrual cycle and contribute to uterine 557 health. Moreover, several gut microbes release short-chain fatty acids (SCFA; e.g., 558 butyrate, acetate and propionate), which participate as anti-inflammatory mediators 559 maintaining gut barrier function and physiological inflammation. When the gut 560 microbiome is disrupted (dysbiosis), the overgrowth of β-glucuronidase-producing 561 bacteria may lead to hyperestrogenic levels commonly reported in different gynecological 562 pathologies. On the other hand, gut dysbiosis can be linked to a reduction of SCFA-563 producing bacteria, resulting in increased pro-inflammatory mediators and systemic 564 inflammation. *Reported β-glucuronidase-producing genera associated with any 565 gynecological disease in case-control studies. (Created with BioRender.com). 566