Microbial regulators of physiological and reproductive health in women of reproductive age: their local, proximal and distal regulatory roles.

Research on the female microbiome has progressed beyond simple microbiome-disease associations to investigating how modulating its composition and functions clarifies causal roles in physiology and reproductive health, and informs preventive and therapeutic strategies. Such interventions include local regulation of the reproductive tract and distal modulation via the gut microbiota. Summarizing their impacts and mechanisms will enhance our understanding of female host-microbe interactions and advance precise, effective microbiome-targeted therapies. Given the pivotal role of the microbiota in reproductive health, an increasing number of studies have attempted to mitigate the detrimental effects of gynecological disorders on reproductive health through the local administration of probiotics. Lactobacillus , as the dominant genus in the female reproductive tract of healthy women, has become the primary candidate for probiotic interventions due to its multifaceted protective mechanisms. Lactobacillus exerts antimicrobial effects by producing H₂O₂, bacteriocin-like substances, biosurfactants (BS), and organic acids 221 , 222 . These metabolites disrupt the formation of pathogenic biofilms, hinder pathogen adhesion to the epithelial surface, and promote microbial competition, thereby limiting the colonization and survival of pathogens such as G. vaginalis , Escherichia coli , and Candida albicans 221 , 223 , 224 (Fig. 4a ). For instance, BSs significantly reduce the formation of pathogenic biofilms by interfering with pathogen adhesion and enhance the ecological competitiveness of Lactobacillus in the vaginal microenvironment 221 , 225 , 226 . Additionally, organic acids produced by Lactobacillus lower vaginal pH, creating an inhospitable environment for opportunistic pathogens 24 . Beyond antimicrobial activity, Lactobacillus also contributes to maintaining the integrity of the genital mucosal barrier. A cervicovaginal mucus layer dominated by L. crispatus serves as an effective physical and biological barrier against HIV invasion 227 , 228 . L. crispatus has been shown to inhibit the expression of sialidase in G. vaginalis , thereby preventing epithelial cell degradation and preserving the mucus barrier 229 (Fig. 4a ). Collectively, these findings highlight the dual role of Lactobacillus in pathogen exclusion and mucosal homeostasis. Fig. 4 In situ probiotic supplementation improves the pathological state of the female reproductive tract through local and proximal regulation. a The possible roles of probiotics supplemented in the vagina. They inhibit the growth of pathogens and destroy biofilms through their own metabolites (I). In the process, the inflammatory response caused by pathogens is reduced (II). By inhibiting the expression of glycosyl chain-degrading enzymes by pathogens, the barrier function of the vaginal mucosa is enhanced or restored, and the occurrence of sexually transmitted diseases (Human immunodeficiency virus, HIV) and UGT infections is prevented (III). b Probiotic regulate the immune environment of the uterine cavity. They reduce inflammation and fibrosis after endometrial damage by regulating the polarization of macrophages to M2. The figure was created with BioRender.com. a The possible roles of probiotics supplemented in the vagina. They inhibit the growth of pathogens and destroy biofilms through their own metabolites (I). In the process, the inflammatory response caused by pathogens is reduced (II). By inhibiting the expression of glycosyl chain-degrading enzymes by pathogens, the barrier function of the vaginal mucosa is enhanced or restored, and the occurrence of sexually transmitted diseases (Human immunodeficiency virus, HIV) and UGT infections is prevented (III). b Probiotic regulate the immune environment of the uterine cavity. They reduce inflammation and fibrosis after endometrial damage by regulating the polarization of macrophages to M2. The figure was created with BioRender.com. Certain pathogens associated with reproductive tract inflammation induce the release of pro-inflammatory cytokines (e.g., IL-1β, IL-6, IL-8) and activate the NF-κB signaling pathway, thereby amplifying the inflammatory response 230 – 232 . Evidences indicated that L. crispatus in the vagina, as well as intravaginal administration of probiotic strain like Lactobacillus johnsonii HY7042, L. crispatus CTV-05, L. crispatus G-7 counteracted these effects by regulating the expression of inflammatory cytokines and/or suppressing NF-κB activation, thereby mitigating pathogen-induced immune responses and contributing to local immune homeostasis 233 – 237 . These findings suggest that, beyond their direct antimicrobial activity, Lactobacillus modulates host immune responses by mitigating pathogen-induced inflammation, thereby contributing to the restoration of vaginal microbial homeostasis (Fig. 4a ). Although clinical evidences have demonstrated the beneficial effects of probiotics in ameliorating CE, including modulation of the endometrial microbiota and improved pregnancy outcomes, the underlying mechanisms remain insufficiently defined 238 , 239 . The latest study revealed that vaginal administration of Lactobacillus exerted a beneficial reparative effect on the damaged endometrium in rats. This effect is achieved by modulating the local immune environment, particularly through macrophage polarization, which helps alleviate inflammation and fibrosis, thereby facilitating tissue regeneration and recovery of the endometrium 240 (Fig. 4b ). Changes in the immune environment of the endometrium further regulate endometrial receptivity and decidualization 241 . In CE, the CD4 + T cell profile in the endometrium is significantly altered, including the ratio of Th1, Th2, Foxp3 + Treg and Th17 242 – 244 . In particular, the increased abundance of pro-inflammatory Th1 and Th17 cells has been associated with impaired embryo implantation and reduced implantation success 244 , 245 . Strains in most probiotic supplements such as Lactobacillus rhamnosus GG, Bifidobacterium adolescentis have demonstrated the capacity to modulate T cells. Notably, these strains improve pathological conditions by altering the Th1/Th2 or Th17/Treg balance 246 – 248 . These findings enlighten us to consider probiotic supplementation to maintain or improve pregnancy success in women. To fully harness these benefits, future studies should aim to identify specific probiotic strains with potent immunomodulatory effects on the endometrium and elucidating the molecular mechanisms, particularly their influence on T cell subsets such as Th1, Th2, Th17, and Treg. In addition, it is necessary to define the optimal timing, dosage, and delivery route of probiotic administration to achieve precise and effective immune modulation. These studies may provide valuable insights for developing microbiota-based interventions to enhance endometrial health and improve reproductive outcomes. Expanding the scope of probiotic supplementation beyond lactobacilli to provide more options for preventing and improving women’s health would be very promising and meaningful. “Not all or only Lactobacillus are beneficial to women’s health”, provides us with insight 39 . Indeed, several other lactic acid bacteria like Lactococcus sp ., Bifidobacterium sp ., Enterococcus sp ., and Streptococcus sp . were also partially detected in the vagina 233 , 249 – 251 . Vaginal Bifidobacterium , particularly Bifidobacterium longum , possess lactic acid-producing capability comparable to those of L. crispatus 252 , suggesting a potential protective role in maintaining vaginal pH stability. Moreover, studies have shown that oral administration of B. longum NK49 or Bifidobacterium bifidum modulated the host immune response to Gardnerella -induced vaginosis by inhibiting activation of the NF-κB pathway and reducing the expression of pro-inflammatory cytokines 253 , 254 , highlighting the potential of some non- Lactobacillus lactic acid-producing strains to improve female physiological health. But the comprehensive functional and safety data remain limited. Certain lactic acid-producing bacteria that coexist at low abundance within the healthy vaginal microbiota have frequently been associated with adverse gynecological outcomes 29 , 251 . L. iners strains typically exhibit limited lactic acid production, weak epithelial adhesion, and the expression of virulence-associated proteins, all of which may compromise their ability to inhibit pathogens and potentially trigger inflammation 255 . Therefore, while non- Lactobacillus lactic acid-producing bacteria may be considered potential probiotic candidates, rigorous evaluation is essential. Such assessments should include 256 : (1) genomic features, including the presence of genes encoding antimicrobial substances (e.g., bacteriocins, H₂O₂), and the absence of pathogenicity or toxin-related genes; (2) a clear antibiotic resistance profile, ensuring the absence of mobile resistance genes (e.g., erm , tet , van ); (3) immunomodulatory properties; (4) lactic acid production and pH-lowering capacity, especially the production of D-lactic acid, which has stronger inhibitory effects on vaginal pathogens and mucosal protection than L-lactic acid 255 ; and (5) the potential to produce biogenic amines; (6) There should be no functional spillover beyond the scope of the targeted disease. A comprehensive focus on both safety and functionality is critical for the selection of next-generation probiotics. Although in situ interventions targeting the reproductive system remain important, an increasing number of studies emphasize that the gut is a key site for distal regulation of female physiological processes. This is inseparable from the pivotal role of the gut microbiota and its metabolites. Targeted modulation of the female gut microbiota can effectively maintain and improve the microbial composition. Microbial metabolites, such as short-chain fatty acids (SCFAs), bile acids (BAs), and branched-chain amino acids (BCAAs), have the potential to regulate reproductive health. Therefore, we focus on gut microbial metabolites. Importantly, elucidating the underlying pathways of these metabolites, as well as their possible limitations or ambiguities, can provide valuable insights for subsequent gut microbiota-based regulation of female reproductive health. SCFAs are the primary metabolites of gut microbiota and are crucial for both intestinal and systemic physiological functions. Modulating SCFAs levels helps improve metabolic abnormalities associated with certain female diseases such as PCOS 257 , 258 . In women with PCOS, there’s a noticeable decrease in gut bacteria like Faecalibacterium , Akkermenisa , and Bifidobacterium 219 , 259 – 263 . This reduction is directly linked to significantly lower levels of SCFAs, particularly butyrate 264 . Bifibacterium , Faecalibacterium , Butyricimonas , and Akkermansia are recognized as primary producers of SCFAs 265 , 266 . The therapeutic potential of gut microbiota-derived SCFAs extends beyond PCOS. They have also shown beneficial effects in other gynecological conditions, including endometriosis and gynecological cancers like cervical and ovarian cancer 267 , 268 . Therefore, it is crucial to either increase the diversity of SCFA-producing microbes or specifically promote the growth and activity of SCFAs producers, especially those that generate butyrate for preventing or alleviating metabolic or immune issues stemming from estrogen dysregulation. Glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) are involved in regulating systemic glucose homeostasis, including insulin secretion 269 – 271 . Both have been noted in numerous studies on PCOS. The link between SCFAs and glucose metabolism is revealed by the fact that increased levels of butyrate produced by gut microbiota significantly promote the release of GLP. SCFAs, by activating G protein-coupled receptors (GPRs) that act as free fatty acid receptors 272 , promote the secretion of key gut hormones such as GLP-1, GIP, and cholecystokinin (CCK) 273 , 274 (Fig. 5Ⅰ ). This process helps regulate metabolic abnormalities in endocrine disorders, offering a beneficial effect on the host. Furthermore, SCFAs can also participate in metabolic homeostasis and even inflammation regulation by enhancing the integrity of the intestinal barrier, reducing the translocation of bacterial endotoxins across the intestinal wall, and autoinhibiting histone deacetylase (HDAC) 60 , 268 . Fig. 5 Possible mechanisms of probiotics regulating intestinal microbiome and its metabolites to improve women’s physiological and reproductive health. (Ⅰ) Short-chain fatty acids (SCFAs) bind to G protein-coupled receptors (GPRs) on intestinal cells, promote the release of hormones such as Glucagon-like peptide-1 (GLP-1) and further participate in regulating glucose homeostasis, thereby alleviating polycystic ovary syndrome (PCOS)-related metabolic abnormalities. (Ⅱ) Bile acids (BAs) and SCFAs play similar roles in regulating glucose homeostasis. The gut microbiota regulates the composition of the BAs pool by producing key enzymes such as bile salt hydrolases (BSHs). Secondary bile acids generated through these enzymatic processes induce T cell differentiation and promote the secretion of IL-22 by innate lymphoid cells (ILCs), thereby indirectly improving ovarian dysfunction and maintaining pregnancy health. The influence of BAs on T cell differentiation has also been noted in probiotics, which are involved in the induction and regulation of regulatory T cells (Tregs) (gray line). Probiotics induce Tregs by activating tolerogenic dendritic cells (DCs) via Toll-like receptors (TLRs) and by activating Treg-associated molecules, including interleukin (IL)-10, transforming growth factor (TGF)-β, and Forkhead box P3 (FoxP3), thereby enhancing immune tolerance and exerting anti-inflammatory effects. Linking probiotics, BAs, and T cells may open new avenues for developing microbiome-targeted interventions to enhance female reproductive health. (Ⅲ) Moderate branched-chain amino acids (BCAAs) inhibit the continuous activation of mTOR signaling on insulin receptor IRS-1, promote the coupling of insulin to the insulin receptors, thereby alleviating insulin resistance and regulating blood sugar. Appropriate amounts of BCAAs also have a positive effect on inducing trophectoderm movement, driving embryo implantation and early fetal development in pregnant women. (Ⅳ) Fluctuations in circulating estrogen levels modulate estrogen and progesterone signaling responses in the reproductive tract, thereby influencing uterine function and fertility. The figure was created with BioRender.com. (Ⅰ) Short-chain fatty acids (SCFAs) bind to G protein-coupled receptors (GPRs) on intestinal cells, promote the release of hormones such as Glucagon-like peptide-1 (GLP-1) and further participate in regulating glucose homeostasis, thereby alleviating polycystic ovary syndrome (PCOS)-related metabolic abnormalities. (Ⅱ) Bile acids (BAs) and SCFAs play similar roles in regulating glucose homeostasis. The gut microbiota regulates the composition of the BAs pool by producing key enzymes such as bile salt hydrolases (BSHs). Secondary bile acids generated through these enzymatic processes induce T cell differentiation and promote the secretion of IL-22 by innate lymphoid cells (ILCs), thereby indirectly improving ovarian dysfunction and maintaining pregnancy health. The influence of BAs on T cell differentiation has also been noted in probiotics, which are involved in the induction and regulation of regulatory T cells (Tregs) (gray line). Probiotics induce Tregs by activating tolerogenic dendritic cells (DCs) via Toll-like receptors (TLRs) and by activating Treg-associated molecules, including interleukin (IL)-10, transforming growth factor (TGF)-β, and Forkhead box P3 (FoxP3), thereby enhancing immune tolerance and exerting anti-inflammatory effects. Linking probiotics, BAs, and T cells may open new avenues for developing microbiome-targeted interventions to enhance female reproductive health. (Ⅲ) Moderate branched-chain amino acids (BCAAs) inhibit the continuous activation of mTOR signaling on insulin receptor IRS-1, promote the coupling of insulin to the insulin receptors, thereby alleviating insulin resistance and regulating blood sugar. Appropriate amounts of BCAAs also have a positive effect on inducing trophectoderm movement, driving embryo implantation and early fetal development in pregnant women. (Ⅳ) Fluctuations in circulating estrogen levels modulate estrogen and progesterone signaling responses in the reproductive tract, thereby influencing uterine function and fertility. The figure was created with BioRender.com. During pregnancy, alterations in SCFAs derived from gut microbiota indirectly contribute to maintaining maternal metabolic homeostasis, supporting reproductive function, and promoting fetal development 275 – 277 . Elevated intestinal SCFA levels during pregnancy have been associated with a reduced risk of inflammation-related spontaneous preterm birth, likely by preserving intestinal epithelial integrity and attenuating the delivery pathway triggered by bacterial translocation and endotoxin-induced inflammatory mediators and prostaglandins 275 , 278 . Abnormal SCFAs metabolic profiles contribute to the development and progression of gestational diabetes mellitus (GDM), preeclampsia (PE), and intrahepatic cholestasis of pregnancy (ICP) 279 . Notably, Specific probiotic strains effectively reduce the risk of GDM and PE, possibly by restoring microbial balance and modulating SCFA-mediated pathways that reinforce the gut barrier, and suppress the production of pro-inflammatory cytokines 60 , 280 , 281 , highlighting the critical mediating role of SCFAs during pregnancy. These findings further suggest that intestinal SCFA levels may hold significant potential for preventing or ameliorating female reproductive health, including adverse pregnancy outcomes, by modulating metabolic and immune pathways. Although SCFAs are beneficial to metabolic health, they also provide energy to the host and therefore may contribute to increased weight gain 282 . Therefore, when modulating the gut microbiota to alter SCFA levels, it is crucial to simultaneously consider their bioavailability at both the local intestinal and systemic levels to avoid the metabolic burden caused by excess energy. Moreover, it is advisable to selectively promote the production of those SCFAs beneficial to the host based on individual metabolic profiles, while minimizing the overproduction of those that may induce metabolic stress. The gut microbiota play a pivotal role in shaping the BAs pool by mediating their transformation and modification, influencing their production, and modulating BA receptor signaling pathways. Microbial intervention strategies have been shown to effectively regulate BA metabolism 283 , 284 . For example, Lactobacillus reuteri J1 has been shown to significantly increase levels of ursodeoxycholic acid (UDCA) and lithocholic acid (LCA), two secondary BAs known to play an important role in host metabolic regulation 284 . Elevated BA pools have been associated with increased abundances of specific bacterial taxa, including Lactobacillus , Bifidobacterium , Akkermansia , Bacteroides , Faecalibacterium prausnitzii , and Clostridium 283 , 284 . This association is largely driven by microbial bile salt hydrolases (BSHs), which deconjugates conjugated BAs, generating free BAs that serve as substrates for subsequent dehydroxylation reactions 283 , 285 – 289 (Fig. 5Ⅱ ). Notably, certain Clostridium also express 7α-dehydroxylation activity, catalyzing the conversion of primary BAs into secondary BAs such as deoxycholic acid (DCA) and LCA 290 , 291 . These secondary BAs can profoundly influence BAs receptor signaling and immune modulation. For example, knockdown of intestinal commensal bacteria that produce BAs ( Bacteroides thetaiotaomicron and Bacteroides fragilis ) inhibits their ability to induce and number of Treg cells 292 . BAs are increasingly recognized for their role in modulating T cell subsets, which impacts female reproductive health. Specifically, certain BA derivatives, like 3-oxoLCA and isoalloLCA, reduced Th17 cell differentiation by directly binding to the key transcription factor RORγt or promoted Treg differentiation by inducing mitochondrial reactive oxygen species (ROS) production 293 (Fig. 5Ⅱ ). Regulation of the Th17/Treg ratio is not restricted to the gut; in the endometrium, T cells differentiation is critical for successful embryo implantation 294 . During healthy pregnancy, a Th17/Treg ratio skewed toward Treg cells favors maternal immune tolerance to the fetus by attenuating inflammatory responses at the maternal-fetal interface (e.g., reduced IL-6 levels) and protecting against unexplained pregnancy loss, such as recurrent spontaneous abortion (RSA) 295 , 296 . Although the precise mechanisms by which BAs modulate immune responses at the maternal-fetal interface remain unclear, emerging clinical evidence links abnormal serum total BA levels to pregnancy complications, including GDM and ICP 289 , 297 , 298 . Furthermore, BAs regulate IL-22 production by engaging G protein-coupled receptor 5 (TGR5), a G protein-coupled bile acid receptor expressed on intestinal T cells and innate lymphoid cells, thereby activating the GATA3 signaling pathway 187 . IL-22, in turn, alleviates ovarian dysfunction in PCOS by modifying ovarian features such as the number of cystic follicles and corpora lutea 187 (Fig. 5Ⅱ ). Moreover, similar to SCFAs, BAs promote GLP-1 secretion by intestinal L cells, suppress inflammation, and participate in the regulation of female reproductive health 299 (Fig. 5Ⅱ ). Collectively, these observations support the hypothesis that BAs act not only as metabolic regulators but also as key mediators of female reproductive health and pregnancy outcomes. These findings further encourage the exploration of the potential to “modulate the gut microbiota to reshape BA composition, thereby mediating immune responses and improving female reproductive health.” Although current studies indicate that certain bacterial strains can influence specific immune cell populations such as the induction and regulation of Treg cells by probiotics 248 , 300 – 302 (Fig. 5Ⅱ ), future research should focus on integrating the probiotic-BAs-T cells regulatory axis. Given the pivotal role of T cells in female conception and emerging evidence that BAs modulate T cells differentiation, such an approach may open new avenues for developing microbiota-targeted interventions to enhance female reproductive health. To advance this field, it will be essential to determine the dependency between BAs and their downstream receptors, identify the pathways through which they mediate immune responses, and observe the resulting immune cell differentiation trends, in order to elucidate the mechanisms by which BAs serve as a bridge between immune function and reproductive health. Moreover, significant variations in BSHs and 7α-dehydroxylase activity may exist among bacterial strains, even within the same genus. Therefore, future studies should prioritize the screening and identification of functional strains to determine their specific roles in BAs metabolism and establish a link with reproductive health. Insufficient BCAAs lead to abnormalities in follicular development, angiogenesis, and sex hormone synthesis driven by elevated ROS levels, thereby disrupting normal reproductive processes 207 , 303 – 305 . Interestingly, BCAA dietary supplementation prevents ROS-induced POI in female mice 304 . These findings prompt further exploration of microbiota-derived BCAAs as potential gut targets in the regulation of female reproductive capacity. Emerging evidence indicates that certain gut microbial taxa are closely involved in BCAA metabolism, including Prevotella copri , Bacteroides spp ., Escherichia coli , Ruminococcus gnavus , F. prausnitzii , Parabacteroides merdae , Butyrivibrio crossotus , and Eubacterium siraeum 306 – 310 . In particular, increased abundance of P. copri and Bacteroides spp . is associated with enhanced endogenous BCAA biosynthesis, as these microbes encode key enzymes such as d-citramalate synthase (involved in isoleucine synthesis) and isopropylmalate (IPM) synthase (critical for leucine synthesis) 310 , 311 . In contrast, F. prausnitzii , P. merdae , and R. gnavus primarily mediate BCAA catabolism, with the genome of F. prausnitzii notably enriched in genes encoding BCAA inward transporters 308 , 312 , 313 . Furthermore, gut microbiota remodeling drives metabolic reprogramming of BCAAs 310 , 312 , a phenomenon experimentally confirmed in specific Lactobacillus spp . strains 314 , 315 . BCAAs are essential for multiple aspects of female reproduction, ranging from embryo implantation to fetal growth. These processes appear to be dependent on the involvement of the mTOR signaling pathway. By activating mTOR, BCAAs enhance the motility of the trophectoderm, a critical step for blastocyst activation and successful embryo implantation 304 , 316 – 319 (Fig. 5Ⅲ ). During subsequent fetal development, BCAAs can also promote protein synthesis or stimulate the hepatic secretion of hormones such as insulin-like growth factor-I (IGF-I) and IGF-II through the mTOR pathway, thereby supporting fetal growth 320 – 322 (Fig. 5Ⅲ ). Additionally, BCAAs seem to maintain and improve ovarian function 322 . Although the precise mechanisms remain under investigation, it is hypothesized that this effect is also mediated via the mTOR pathway, which is known to regulate key ovarian processes including autophagy in granulosa cells, follicle activation, and maintenance of the follicular pool 323 – 327 . These findings indicate that BCAAs play multifaceted roles in female reproductive health by influencing ovarian function, embryo implantation, and early fetal development. The mTOR pathway acts as a central regulator in these processes and warrants further research to fully elucidate this relationship. The role of BCAAs is a subject of ongoing debate. While high levels of BCAAs are strongly linked to insulin resistance 328 , a moderate amount of BCAAs restore the suppressive effect of the mTOR pathway on insulin signaling, thereby promoting insulin secretion 329 , 330 . Insulin activates the PI3K/Akt signaling pathway, which regulates autophagy, inflammation, and oxidative stress in ovarian granulosa cells, thereby promoting follicular development and improving ovarian dysfunction 331 – 335 . This suggests that BCAAs indirectly influence ovarian function by modulating the insulin signaling pathway. Current research has not yet directly established a relationship between BCAA microbiota and infertility. However, preliminary evidence suggests a potential link between the gut microbiota, BCAA metabolism, and female reproductive health. Future research should investigate BCAA levels, the composition and function of related bacteria in the gut of infertile patients. It is also important to develop BCAA-modulating interventions, such as probiotics, and study how they affect BCAA-producing bacteria, substrate utilization, gene expression, and enzyme activity. Crucially, future studies must evaluate the effects of BCAA intervention in a bidirectional manner, because different pathological states can lead to vastly different BCAA levels, and both excessively high or low levels alter ovarian or insulin function. Estrogen levels in healthy women fluctuate regularly with the menstrual cycle, rising during the follicular phase, reaching a peak during ovulation, and then decreasing due to increased progesterone levels, preparing for embryo implantation and conception 27 , 111 . However, the maintenance of this hormonal dynamic balance is inseparable from the participation of the intestinal microbiome. Microorganisms such as Escherichia and Bacteroides fragilis that express β-glucuronidase influence circulating estrogen levels 259 , 336 , 337 . Fluctuations in estrogen disrupt the coordination between estrogen and progesterone signaling within the uterine environment, affecting endometrial receptivity to embryo implantation 338 , 339 (Fig. 5Ⅳ ). SCFAs produced by gut microbes also have the capacity to regulate hormone secretion along the HPO axis 340 , 341 , similar to the estrobolome. Microbial interventions that enhance SCFA production have been shown to effectively stimulate gut hormone release 262 , 315 , 342 , 343 . Among these, peptide YY (PYY) plays a critical role by promoting the secretion of LH and FSH, thereby participating in the neuroendocrine regulation of the HPO axis 262 , 340 , 344 . These findings underscore the pivotal role of microbial signals in maintaining sex hormone homeostasis. In this context, intestinal microbial metabolites directly or indirectly act on the intestinal endocrine system, cross the intestinal barrier and enter the host circulation, forming an important bridge between the microbiome and female reproductive health. Modulating the gut microbiota not only helps alleviate female endocrine disorders and ovarian diseases, but also provides us with new directions and possibilities for further thinking about how to promote female reproductive health through microbial-mediated estrogen regulation strategies.

Estrogen plays a pivotal role in female reproductive physiology by regulating the HPO axis, maintaining endometrial receptivity, and supporting ovulatory function 186 . Growing evidence suggests that the gut microbiota not only regulates host metabolism and immunity, but also communicates with the reproductive endocrine system through modulation of estrogen metabolism 116 , 187 . In this context, the concept of the “estrobolome”, defined as the collection of gut microbial genes capable of metabolizing estrogens, has emerged 188 – 192 . Elucidating how gut microbes contribute to the development of estrogen imbalance-related disorders through microbe-derived signaling molecules, metabolic pathways, or immunomodulatory mechanisms not only provides a mechanistic basis for distal microbial regulation, but also updates our understanding of the microbiome’s role in female physiology and reproductive health. In the human estrobolome, β-glucuronidase, a member of the glycoside hydrolase 2 family (GH2), plays a central role in estrogen metabolism. This enzyme family also includes β-mannosidases, and β-galactosidases, which, like β-glucuronidase, are predominantly encoded and produced by the gut microbiota 193 , 194 . Both β-glucuronidase and β-galactosidase are involved in the biotransformation of estrogens in humans 195 . β-glucuronidase is particularly important for the deconjugation and reactivation of estrogens within the intestinal environment 193 , 196 (Fig. 3 ). An increase in the expression of β-glucuronidase in the gut, or a higher abundance of bacteria harboring this enzymatic activity, can accelerate estrogen deconjugation and subsequent hydroxylation, thereby raising circulating levels of the free estrogens and maintaining at a physiological level 193 . Notably, dysregulation of β-glucuronidase activity has been implicated in several estrogen-driven disorders, including endometriosis, polycystic ovary syndrome (PCOS), and ovarian cancer 192 , 197 , 198 . Currently, a total of 279 β-glucuronidase-related genes have been identified in the human gut, with 93.5% belonging to four major bacterial phyla, predominantly Bacteroidetes (52%) and Firmicutes (43%) 193 , 199 . Alterations in the ratio between Firmicutes and Bacteroidetes can influence overall β-glucuronidase activity 200 , 201 (Fig. 3 ). An increase in Bacteroidetes accompanied by a decrease in Firmicutes is often associated with elevated β-glucuronidase activity in PCOS 192 , 202 . This microbial imbalance has been observed in patients with PCOS and primary ovarian insufficiency (POI) 187 , 203 – 207 . Enhanced β-glucuronidase activity alters the levels of free estrogen, thereby modulating the activity of estrogen receptors (ERα or ERβ) 208 . Excessive ERβ signaling, in particular, has been shown to promote inflammation in endometrial stromal cells 209 , 210 (Fig. 3 ). Additionally, an inverse correlation between β-glucuronidase activity and the abundance of Lactobacilli in the reproductive tract has been reported, suggesting that increased gut-derived estrogenic activity may be linked to dysbiosis of the reproductive tract microbiota 211 . However, this association is currently based primarily on correlative analyses, and definitive causal evidence is lacking. It remains unclear whether elevated β-glucuronidase activity directly drives compositional shifts in the reproductive tract microbiota, or whether such changes are mediated indirectly through alterations in systemic hormone levels. Fig. 3 Distal effects and possible mechanisms of disturbed intestinal microbiota on female fertility. The gut microbiota influences the estrogenome, which refers to the collection of microbial genes in the human gut capable of metabolizing estrogens. Microbially expressed β-glucuronidase plays a particularly important role in the deconjugation and reactivation of estrogens in the gut environment. Both Bacteroidetes and Firmicutes can express β-glucuronidase 193 , 199 . An increased ratio of Firmicutes to Bacteroidetes (F/B) is associated with overall elevated β-glucuronidase activity. Enhanced β-glucuronidase activity alters the levels of free estrogens, thereby modulating the activity of estrogen receptors (ERα or ERβ) and affecting the health of female pregnancy-related sites 208 – 210 . The gut estrogenome imbalance also disrupts the secretion of follicle-stimulating hormone (FSH), luteinizing hormone (LH), or the FSH/LH ratio along the HPO axis 214 , 215 . Furthermore, dysbiosis exacerbates the occurrence and progression of leaky gut by downregulating genes (ZO-1,Occludin) involved in maintaining gut barrier function. Leaky gut facilitates microbial translocation and the entry of microbial metabolites into systemic circulation, triggering local and systemic inflammatory cascades. These microbes or their metabolites induce the production of pro-inflammatory cytokines in epithelial cells via recognition by Toll-like receptors (TLRs) and recruit immune cells, thereby amplifying systemic inflammation and compromising female fertility 60 , 196 , 216 . In addition, Some neuroactive metabolites act on the hypothalamus and indirectly influence hormonal regulation via the hypothalamic-pituitary-ovarian (HPO) axis 196 , 220 . The figure was created with BioRender.com. The gut microbiota influences the estrogenome, which refers to the collection of microbial genes in the human gut capable of metabolizing estrogens. Microbially expressed β-glucuronidase plays a particularly important role in the deconjugation and reactivation of estrogens in the gut environment. Both Bacteroidetes and Firmicutes can express β-glucuronidase 193 , 199 . An increased ratio of Firmicutes to Bacteroidetes (F/B) is associated with overall elevated β-glucuronidase activity. Enhanced β-glucuronidase activity alters the levels of free estrogens, thereby modulating the activity of estrogen receptors (ERα or ERβ) and affecting the health of female pregnancy-related sites 208 – 210 . The gut estrogenome imbalance also disrupts the secretion of follicle-stimulating hormone (FSH), luteinizing hormone (LH), or the FSH/LH ratio along the HPO axis 214 , 215 . Furthermore, dysbiosis exacerbates the occurrence and progression of leaky gut by downregulating genes (ZO-1,Occludin) involved in maintaining gut barrier function. Leaky gut facilitates microbial translocation and the entry of microbial metabolites into systemic circulation, triggering local and systemic inflammatory cascades. These microbes or their metabolites induce the production of pro-inflammatory cytokines in epithelial cells via recognition by Toll-like receptors (TLRs) and recruit immune cells, thereby amplifying systemic inflammation and compromising female fertility 60 , 196 , 216 . In addition, Some neuroactive metabolites act on the hypothalamus and indirectly influence hormonal regulation via the hypothalamic-pituitary-ovarian (HPO) axis 196 , 220 . The figure was created with BioRender.com. The HPO axis is involved in the regulation of circulating estrogen levels 212 . The HPO axis is initiated by the secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus, which acts on the pituitary gland 196 . In response, the anterior pituitary releases two key gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which target the ovaries to regulate the cyclical fluctuations of estrogen and progesterone 196 . Dysregulation of this axis leads to ovarian dysfunction and abnormal estrogen secretion, which can directly impair fertility by altering the thickness and quality of the endometrium 196 , 212 , 213 . Such imbalances within the intestinal estrobolome interfere with the normal negative feedback regulation of the HPO axis, partially explaining the abnormal secretion patterns of FSH, LH, or the FSH/LH ratio observed in estrogen imbalance-related disorders 214 , 215 (Fig. 3 ). These findings underscore the essential role of the estrobolome in maintaining systemic estrogen homeostasis. Although changes in β-glucuronidase activity highlight the close relationship between the gut microbiota and reproductive endocrine function, the specific role of gut-derived β-glucuronidase in the regulation of the HPO axis remains to be elucidated. This includes the precise identification of microbial taxa responsible for β-glucuronidase production, quantification of enzyme activity, and evaluation of its association with hormonal dynamics along the HPO axis. In addition to promoting endocrine disorders, an imbalance in the gut microbiota exacerbates inflammatory responses and metabolic abnormalities. Dysbiosis-induced increases in intestinal permeability facilitate microbial translocation and the entry of microbial metabolites into systemic circulation, thereby initiating both local and systemic inflammatory cascades 60 , 196 . For instance, LPS activates macrophages and induces the production of pro-inflammatory cytokines such as TNF-α and IL-6 216 (Fig. 3 ). These inflammatory mediators not only disturb metabolic homeostasis but also influence reproductive function. TNF-α impairs insulin signaling by activating JNK1 and NF-κB pathways, contributing to insulin resistance, a hallmark of PCOS 216 – 218 . In parallel, IL-6 upregulates the expression of CYP17A1, a key enzyme in androgen synthesis 219 , thereby linking inflammation to ovarian hormone production. Moreover, gut microbiota composition modulates immunoregulatory factors, notably IL-22. The reduction of IL-22 exacerbates the inflammatory microenvironment in ovarian granulosa cells and may contribute to the autoimmune abnormalities observed in POI 187 , 214 . Specific microbial taxa, such as secondary bile acid-producing bacteria and reproductive tract-associated Lactobacillus spp ., are known to regulate IL-22 expression 187 , 214 . Notably, both groups are depleted in estrogen-related disorders, including PCOS and POI. This strongly suggests that characteristic changes in the female gut microbiota have an important impact on female reproductive health. The role played by the gut microbiome in the pathology of endocrine and metabolic disruption is comprehensive and complex. Such complexity dictates that it remains challenging to truly elucidate the mechanisms behind distal regulation. Future research should focus on how the gut microbiota regulates the structure and function of the reproductive tract microbiota through hormones, metabolism, and the immune system, especially examining whether differences exist in various physiological and pathological states (such as pregnancy, gynecological diseases, etc.). In addition to identifying microbial taxa involved in estrogen metabolism, attention should also be given to other gut microbiota-derived metabolites. Certain neuroactive compounds directly activate hypothalamic neurons, including GnRH neurons 196 , 220 (Fig. 3 ), thereby influencing endocrine regulation. Elucidating these processes will help uncover the mechanisms underlying gut–reproductive tract microbial interactions and provide a more comprehensive understanding of the precise role of the gut–reproductive axis in female physiology and reproductive health.

A small number of clinical human trials have demonstrated the beneficial effects of probiotic in situ therapy on reproductive tract health. For example, vaginal administration of a probiotic mixture has been shown to significantly reduce the recurrence of BV following standard treatment 345 . Some other probiotic strains have shown potential in the management and prevention of vaginosis 346 – 348 . In addition to their established benefits in the LGT, vaginal probiotics have shown promising efficacy in the management of UGT conditions. L. crispatus chen 01 enhanced pregnancy rates in CE patients by improving progesterone levels and endometrial pathology 239 . Based on the influence of the mainstream idea that probiotics regulate the microbiome, some other strategies including prebiotics, synbiotics, postbiotics (non-viable bacterial products or metabolic byproducts from probiotics that confer health benefits to the host) and vaginal microbiota transplantation (VMT) are also being increasingly explored and applied in the clinic. Prebiotics are a class of non digestible dietary ingredients that can selectively promote the growth and metabolism of beneficial bacteria, such as Bifidobacteria and Lactobacili 349 , 350 . Common prebiotics mainly include oligosaccharide carbohydrates such as fructo-oligosaccharide and gluco-oligosaccharide 351 . Recent studies suggest prebiotics also benefit female reproductive health. In a randomized controlled trial involving 42 patients with BV who had received antibiotic treatment, gluco-oligosaccharide vaginal gel significantly reduced Nugent scores (a Gram stain-based scoring system for the diagnosis of BV) and supported restoration of normal vaginal microbiota, thereby reducing the risk of recurrence 352 . Additionally, topical prebiotics contribute to enhancing the recovery of cervical epithelial changes caused by HPV infection and potentially reduce the risk of subsequent development of cervical intraepithelial neoplasia (CIN) 353 – 357 . It is worth noting that although prebiotics have been reported to promote the growth of Lactobacillus and thereby improve vaginal health, the ability of these bacteria to utilize prebiotics is highly strain specific and substrate specific. Therefore, intervention strategies must be tailored to specific microbial characteristics to achieve symptom relief, pathogen suppression, balanced microbiota, HPV clearance, and CIN prevention. In addition, the application of prebiotics in clinical practice of women’s physiology may reduce concerns about the activity and safety of using live bacterial biopharmaceuticals, but their efficacy in in situ interventions remains to be demonstrated across a wider range of cases and populations. Unlike the indirect effects of probiotics or prebiotics, VMT aims to build a new vaginal microecological balance through transferring the entire vaginal community from the vaginal secretions of healthy women to the recipient 358 . Originally investigated for the treatment of BV 359 , 360 , VMT is now also being explored as a potential therapeutic strategy for other conditions such as vulvovaginal candidiasis, endometriosis and gynecological cancers 361 – 363 . Some BV individuals have long-term improvements in vaginal health by VMT, including symptom relief, normalization of Amsel’s criteria, improvement in the vaginal fluid appearance, and restoration of a Lactobacillus -dominated microbial structure 364 , 365 . A recent proof-of-concept study further demonstrated, for the first time, that VMT without antibiotic preconditioning can achieve stable engraftment of donor microbiota, shift the dysbiotic state dominated by Gardnerella to L. crispatus -dominated community, and increase live birth rates in women with recurrent pregnancy loss 365 . This strengthens the potential of VMT to impact related reproductive issues and triggers in-depth consideration of future donor selection strategies. To improve engraftment success and microbiota stability, some researchers have proposed using in vitro competition assays between donor and recipient microbiota to identify optimal donors 365 . Future studies may also simulate the vaginal microenvironment by modulating factors such as pH and nutrient conditions to evaluate donor strain viability and competitive advantage with recipient microbiota. In addition, the interaction between transplanted microbes and host immune responses warrants closer investigation. Despite promising preliminary results, current studies are constrained by small sample sizes and potential confounding factors affecting pregnancy outcomes, necessitating further validation in larger, controlled clinical trials. Moreover, the implementation of VMT faces several challenges, including biosafety concerns, ethical issues, and the lack of standardized protocols for donor screening and microbial preparation. Given the influence of host-specific and racial differences in vaginal microbiota composition, the development of personalized VMT approaches may be essential for optimizing therapeutic outcomes and promoting women’s reproductive and physiological health. In addition to in situ interventions, the regulation of the female reproductive tract by the distal gut microbiota has emerged as a promising area of clinical exploration. Oral microbiota-targeted strategies, such as probiotics, prebiotics, and synbiotics (a synergistic combination of probiotics and prebiotics), have shown beneficial effects in multiple aspects, including reducing the recurrence of BV, optimizing vaginal microbiota composition, increasing Lactobacillus abundance, and improving pregnancy and neonatal outcomes such as pregnancy duration, the rates of cesarean section, newborns’ hospitalization 366 – 371 . Synbiotics are particularly designed to enhance the survival, colonization, and activity of beneficial microorganisms by providing both live microbes and their selective substrates, and are increasingly being explored for their enhanced therapeutic potential compared to single interventions. However, the effectiveness of these approaches is influenced by multiple factors, including inter-individual variability, microbial strain specificity and origin, as well as dosage and duration of administration 372 . Importantly, the influence of the gut–vagina axis extends beyond local vaginal health and involves broader regulatory pathways, including hormonal modulation, systemic metabolic regulation, and immune responses 346 . Clinical studies have demonstrated that probiotic supplementations with different courses significantly reduce markers of inflammation and metabolic dysfunction associated with PCOS 346 , 373 – 378 . Notably, improvements in endocrine parameters, such as elevated sex hormone-binding globulin (SHBG) levels and reduced total testosterone, have also been observed 375 , 379 , supporting the potential of probiotics in endocrine regulation. Nevertheless, the effects of probiotics on hormone levels are not widespread. Although the gut microbiota is known to harbor microbial taxa involved in estrogen metabolism, current evidence is insufficient to elucidate the mechanisms by which microbial alterations influence estrogen homeostasis, let alone reveal the relationship and regulatory mechanism between probiotics and these potential estrogen-related microorganisms. We have evaluated, to the extent possible, microbiome-related interventions aimed at improving women’s physiological health, specifically probiotics, prebiotics, and VMT, and analyzed their therapeutic potential and inherent limitations. Building upon these insights, we propose potential strategies to enhance their clinical applicability and translational potential. Importantly, advancing these interventions requires a comprehensive understanding of how they interact with host physiology and the microbiome. A key prerequisite for successful clinical translation is the establishment of robust associations between the gut and reproductive tract microbiota and reproductive-related diseases, which will facilitate the identification of precise microbiome-based therapeutic targets. In parallel, it is crucial to investigate the mechanisms by which these interventions modulate key microbial communities, with particular attention to their hormonal, immunological, and metabolic regulatory pathways. Elucidating how such interactions influence specific microbial strains or communities within the reproductive tract will help uncover targeted molecular mechanisms, thereby providing a solid foundation for personalized clinical interventions. Looking ahead, well-designed, large-scale, placebo-controlled, double-blind clinical trials are urgently needed to evaluate the safety, efficacy, and long-term outcomes of these strategies. The key considerations outlined here are expected to inform and support the design and implementation of future microbiome-based interventions aimed at promoting female reproductive health.

The lower genital tract (LGT), comprising the cervix and vagina, harbors a microbiota that plays a crucial role in maintaining reproductive health. Numerous studies have established a strong association between the LGT microbiome and various gynecological and reproductive disorders in women 14 , 22 . The LGT microbiota exhibits low diversity and is predominantly composed of the genus Lactobacillus in healthy women, accounting for approximately 99% and 97% of the vaginal and cervical microbiota, respectively 23 . This predominance is closely related to the accumulation of intracellular glycogen in the vaginal epithelium under estrogen stimulation 24 – 26 . Lactobacillus metabolizes glycogen as a carbon source, fermenting it to produce lactic acid, which acidifies the vaginal environment to a pH of 3.5-4.5 24 . This acidic milieu inhibits the growth of pathogenic microorganisms and helps preserve microbial homeostasis 24 (Fig. 1a ). The vaginal microbiota of reproductive-age women is commonly categorized into five community state types (CSTs) 27 . CSTs I, II, III, and V are each dominated by a single Lactobacillus species ( Lactobacillus crispatus , Lactobacillus gasseri , Lactobacillus iners , and Lactobacillus jensenii , respectively), whereas CST IV is characterized by a diverse mixture of facultative and obligate anaerobes 12 , 28 . Fig. 1 Distribution and composition of reproductive tract microorganisms and their impact on women’s physiological health. a Lactobacillus is mainly distributed in the lower genital tract (LGT). Lactobacillus from the LGT is responsible for breaking down glycogen for growth and maintaining the acidic environment of the vagina, which in turn helps stabilize the microecology. b Lactobacillus iners , which lacks the ability to produce D-lactic acid and hydrogen peroxide (H 2 O 2 ), has the capacity to drive the vaginal microbiota from CST III to a high-diversity CST IV. High-diversity microbiota create a favorable environment for their own survival by producing biogenic amines, and they induce adverse pathological states in the reproductive tract in situ by lowering vaginal pH and activating immune responses. c The microbial biomass of the upper genital tract (UGT), comprising the endometrium, fallopian tubes, and ovaries, is low. Existing data suggest that the microbiota of the UGT exhibit higher microbial diversity than that of the LGT, with an increased proportion of non- Lactobacillus bacteria. However, the normal microbiota of the UGT remain poorly characterized due to the specificity of samples, sampling techniques, and limitations in sequencing methodologies. d Disturbances in the microenvironment of the UGT are influenced by the upward migration of pathogenic microbes from the LGT, which can also carry the cytokines they induce. These harmful microbes and molecules trigger inflammatory responses in the UGT by recruiting lymphocytes, affecting T cell differentiation, and activating adaptive immunity, leading to tissue damage and compromising overall reproductive tract health. e Host environmental factors such as menstruation, unprotected vaginal intercourse, uncontrolled antibiotic use, vaginal douching, pregnancy and parturition drive the shift of the three Lactobacillus -dominated healthy microbiota communities toward L. iners -dominated or dysbiotic communities. The figure was created with BioRender.com. a Lactobacillus is mainly distributed in the lower genital tract (LGT). Lactobacillus from the LGT is responsible for breaking down glycogen for growth and maintaining the acidic environment of the vagina, which in turn helps stabilize the microecology. b Lactobacillus iners , which lacks the ability to produce D-lactic acid and hydrogen peroxide (H 2 O 2 ), has the capacity to drive the vaginal microbiota from CST III to a high-diversity CST IV. High-diversity microbiota create a favorable environment for their own survival by producing biogenic amines, and they induce adverse pathological states in the reproductive tract in situ by lowering vaginal pH and activating immune responses. c The microbial biomass of the upper genital tract (UGT), comprising the endometrium, fallopian tubes, and ovaries, is low. Existing data suggest that the microbiota of the UGT exhibit higher microbial diversity than that of the LGT, with an increased proportion of non- Lactobacillus bacteria. However, the normal microbiota of the UGT remain poorly characterized due to the specificity of samples, sampling techniques, and limitations in sequencing methodologies. d Disturbances in the microenvironment of the UGT are influenced by the upward migration of pathogenic microbes from the LGT, which can also carry the cytokines they induce. These harmful microbes and molecules trigger inflammatory responses in the UGT by recruiting lymphocytes, affecting T cell differentiation, and activating adaptive immunity, leading to tissue damage and compromising overall reproductive tract health. e Host environmental factors such as menstruation, unprotected vaginal intercourse, uncontrolled antibiotic use, vaginal douching, pregnancy and parturition drive the shift of the three Lactobacillus -dominated healthy microbiota communities toward L. iners -dominated or dysbiotic communities. The figure was created with BioRender.com. Not all Lactobacillus spp . are beneficial to the LGT. A notable exception is L. iners , which, unlike other dominant Lactobacillus species, acts as a ‘traitor’ within the vaginal Lactobacillus and negatively impacts vaginal health. This detrimental role may be attributed to its reduced genome size and limited metabolic capacity 29 , 30 . Compared with the genome size of healthy vaginal Lactobacillus spp . (approximately 1.5-2.0 Mb) 31 – 34 , L. iners possesses an unusually small genome (~1.3 Mb), comparable in size to those of human symbionts and parasites 29 , 35 , 36 , suggesting an evolutionary shift toward a host-dependent lifestyle. This genome reduction is indicative of decreased metabolic potential. Consequently, L. iners lacks the ability to produce key antimicrobial compounds such as D-lactic acid and hydrogen peroxide (H₂O₂) 30 , 37 , 38 , which are typically synthesized by other Lactobacillus species. Instead of maintaining homeostasis, L. iners relies on metabolic adaptation to fluctuating host microenvironments 29 , 39 (Fig. 1b ). Studies have found that its genome contains key virulence factor genes encoding iron-sulfur proteins, unique σ-factors and inerolysin 29 . Inerolysin, a pore-forming toxin functionally homologous to vaginolysin produced by Gardnerella vaginalis , may compromise the vaginal mucus layer and weaken host defenses 29 . These characteristics underscore the high ecological niche specificity of L. iners and its role in fostering an environment conducive to the overgrowth of anaerobic bacteria associated with CST IV, including Gardnerella , Atopobium , Prevotella , Sneathia , and Megasphaera . This shift contributes to the transition of the vaginal microbiota from CST III to the dysbiotic CST IV state 29 , 40 . CST IV is widely recognized as a hallmark of vaginal dysbiosis, characterized by a polymicrobial consortium dominated by obligate anaerobic bacteria including G. vaginalis , Prevotella , Atopobium , Peptostreptococcus , and Mobiluncus 41 – 44 . These dysbiotic communities deplete lactic acid and produce various biogenic amines, notably putrescine and cadaverine, which elevate vaginal pH above 4.5 and exacerbate the severity of BV 27 , 45 – 49 . Biogenic amines, responsible for the characteristic malodor of BV, are primarily generated by Dialister spp ., Megasphaera , Mobiluncus and several Prevotella spp . 27 , 46 , 50 . These amines negatively impact the growth dynamics of Lactobacillus , such as lag time, growth rate, and lactic acid production, thereby delaying the re-establishment of a healthy vaginal microbiota 51 , 52 (Fig. 1b ). This paradoxically suggests that biogenic amines, as bacterial metabolites, may also play a role in shaping and maintaining the microbial community. Moreover, CST IV-associated bacteria secrete hydrolytic enzymes such as sialidases that degrade mucins 45 , compromising the integrity of the cervicovaginal mucosal barrier and increasing the risk of microbial translocation and ascending infections 53 . This barrier disruption facilitates pro-inflammatory responses via recognition of microbial pathogen-associated molecular patterns (PAMPs) by Toll-like receptors (TLRs) on vaginal epithelial cells, neutrophils and endocervical antigen-presenting cells (APCs) 54 – 56 (Fig. 1b ). In particular, TLR4 recognizes LPS derived from CST IV-associated bacteria via the CD14-MD-2 complex, thereby activating MyD88-dependent pathways to trigger NF-κB signaling 57 , 58 . This cascade promotes the production of pro-inflammatory cytokines and chemokines and enhances lymphocyte recruitment, thereby exacerbating local inflammation 59 . It is noteworthy that the association between CST IV and adverse vaginal health outcomes is not universally observed across all populations. In women of African, Hispanic, and certain Asian ancestries, CST IV may represent a common and stable vaginal community state 1 , 12 , 60 . CST IV is further divided into IV-A, IV-B, and IV-C. IV-A is dominated by Candidatus Lachnocurva vaginae and G. vaginalis ; IV-B is enriched in Atopobium vaginae and G. vaginalis ; and IV-C is characterized by low abundances of Lactobacillus spp ., G. vaginalis , A. vaginae , and Ca. L. vaginae , with a predominance of diverse facultative and obligate anaerobes 61 . The CST IV subtypes further clarify that IV-A and IV-B are the predominant forms and are associated with elevated vaginal pH and higher Nugent scores in African and Hispanic women, whereas IV-C is less prevalent 61 . These differences suggest that the composition and stability of CSTs are influenced by factors such as ethnicity, lifestyle, and geographic environment 60 . The observed ethnic variation in CSTs distribution also indicates that host genetic variation plays a role in determining susceptibility to specific CSTs. Genome-wide association studies (GWAS) have identified multiple loci related to immune signaling and epithelial barrier function that are associated with particular vaginal microbial features, including CSTs dominated by Lactobacillus spp . or by anaerobic taxa 62 – 64 . Polymorphisms in human leukocyte antigen (HLA) genes located in the major histocompatibility complex (MHC) region have been linked to susceptibility to adverse reproductive tract infection outcomes 65 – 67 . HLA-DRB1/DQB1 gene variants have been linked to human papillomavirus (HPV) clearance 66 , suggesting that they may regulate host immune responses to specific pathogens. Although the precise mechanisms by which specific HLA allelic variants shape the vaginal microbiome remain not fully elucidated, evidence from gut microbiome studies offers potential mechanistic insights 68 , 69 . Similarly, variants in innate immune receptors, including TLR2 and TLR4, alter vaginal bacterial composition, influence the inflammatory milieu, and affect the persistence of BV-associated taxa, thereby indirectly impacting the distribution and stability of CSTs 70 – 73 . Thus, although the current CST classification system has been expanded to include 7 CSTs and 13 sub-CSTs 1 , a comprehensive evaluation of vaginal microbiota should integrate environmental, host, and microbial factors. Based on the dynamic fluctuations of the vaginal microbiota across the menstrual cycle, a novel temporal classification framework called vaginal community dynamics (VCDs) categorizes community transition patterns into four types: (1) the constant eubiotic, (2) the menses-related dysbiotic, (3) the unstable dysbiotic, which changes community states for a short while, and (4) the constant dysbiotic 74 . Menses-related dysbiotic, unstable dysbiotic types and constant dysbiotic are associated with shifts toward CST III or IV, driven by host environmental factors such as menstruation, unprotected vaginal intercourse, uncontrolled antibiotic use, vaginal douching, pregnancy and parturition 27 , 75 , 76 (Fig. 1e ). For example, the vaginal microbiome often shifts from relatively stable CST I and CST II to CST III during menstruation 27 , 39 , 74 , 77 . Notably, although the relative abundance of Lactobacillus spp . fluctuates during these dynamic changes, the overall community function can remain stable. For example, vaginal lactate levels and metabolic activity remained largely unchanged in individuals experiencing shifts in the dominant Lactobacillus sp. . This indicates a certain degree of functional redundancy within the vaginal ecosystem 39 and suggests that specific links exist between community composition and functional potential 78 – 82 (Table 1 ). For example, communities dominated by L. crispatus selectively promote the growth of beneficial bacteria while inhibiting L. iners through the regulation of specific vaginal fatty acids 83 . Therefore, integrating analyses of both microbial composition and function in future vaginal microbiome studies may enable a more precise understanding of the material basis and physiological implications of microbial community changes. Table 1 Overview of the microbial and metabolic profiles of reproductive tract related to women’s physiological and reproductive health Physiological state Bacteria Metabolites Relationship between metabolites and microorganisms / host Sampling method Sequencing method References Health L. gasseri  ↓  L. crispatus  ↓  Streptococcus sp .↑ L. iners  ↑  Lactate↑ Acetate↑ When the Lactobacillus -dominated community changes to a Streptococcus sp .-dominated community during menstruation, the metabolites still showed higher levels of lactic acid. Self-collected mid-vaginal swabs during menses 16S rRNA sequencing of V1-V2 region 39 Health Atopobium  ↑  Prevotella  ↑  Other anaerobes↑ L. iners  ↓  Lactate↓ Acetate↑ Succinate↑ Amino acids Sugars The metabolic output of communities with high biodiversity is more variable over time. Self-collected mid-vaginal swabs during menses 16S rRNA sequencing of V1-V2 region 39 VVC L. iners  ↑  Gardnerella  ↑  L-glutamate↑ DDP↑ Glycogen-related metabolites↑ P. bivia was positively correlated with DDP. L. iners was positively correlated with L-glutamate. These metabolites promoted peptidoglycan synthesis and led to Candida infection. Vaginal discharge Metagenomic sequencing on the DNBSEQ platform 78 VVC Candidiasis albicans  ↑  Linoleic acid↑ Arachidonic acid↑ Phenylalanine↑ Tyrosine↑ Tryptophan↑ Sugar alcohols↑ Linoleic acid act as a pro-inflammatory factor in the response of the vaginal mucosa to C. albicans . L-phenylalanine may contribute to the development of VVC. Samples of vaginal discharge Gram stain 79 BV Prevotella  ↑  Atopobium  ↑  Mycoplasma hominis  ↑  Amines↑ SCFAs↑ Nicotinate↑ Organic acids (Malonate and Acetate)↑ Bacterial participation in amino acid decarboxylation reactions to produce biogenic amines. SCFAs may be involved in the recruitment and activation of the innate immune cells in the female genital tract. Acetate was produced by Prevotella and Mobiluncus spp . Vaginal fluids from the left, central and right upper vaginal vaults qPCR 47 BV Megasphaera sp. type 1  ↑  G. vaginalis  ↑  Eggerthella sp. type 1  ↑  Leptotrichia  ↑  Sneathia spp .↑ Prevotella timonensis  ↑  N-acetylneuraminate↑ 12-HETE↑ Arginine↓ Amines↑ N -acetylputrescine↑ Succinate↑ G. vaginalis can transport and catabolise N-acetylneuraminic acid as a carbon and energy source. BV-associated bacteria convert arachidonic acid to 12-HETE, a biomarker of inflammation. Increased utilization of amino acids by bacteria, as evidenced by high levels of amines and degradation products of amines. Swabs from the lateral vaginal wall qPCR and 16S rRNA sequencing of V3-V4 region 46 BV Lactobacillus ↓ G. vaginalis ↑ Atopobium ↑ Prevotella ↑ Veillonella ↑ M. hominis ↑ SCFAs↑ Organic acids (Succinate, Formate, Fumarate)↑ Biogenic amines↑ Hypoxanthine and xanthine↑ Major metabolic changes of BV-related bacteria Vaginal fluids from the left, central and right upper vaginal vaults qPCR 48 BV L. iners  ↑  Gardnerella  ↑  P. timonensis  ↑  Fannyhessea vaginae  ↑  Histidine↓ Arginine↓ Valine↑ Purine↓ Nicotinate and Nicotinamide↓ Eicosenoic acid↑ Carnitine↑ BV-associated bacteria tend to utilize amino acids as a carbon source. Strong association of valine upregulation with biofilm-forming bacterial species such as Prevotella and Sneathia . Eicosenoic acid, a naturally occurring monounsaturated fatty acid and an immune system stimulator, was found to be highly discriminatory for BV. Swabs from the lateral vaginal wall 16S rRNA sequencing of V3-V4 region and qPCR 49 BV VVC VVC_BV Candida (in VVC_BV and VVC)↑ G. vaginalis and Prevotella (in VVC_BV and BV)↑ Indole↑ Chorismate↑ Aromatic amino acid↑ Coenzyme A biosynthesis pathway related to fatty acid synthesis↑ Tryptophan is a known aromatic amino acid present in bacteria. Prevotella , Chlamydia , and certain bacteria associated with BV possess the ability to degrade tryptophan into indole. Fatty acids in the vagina could trigger elevated levels of inflammatory cytokines in VVC_BV. Vaginal secretions Metagenomic sequencing 80 C. trachomatis VVC BV Faecalibacterium  ↑  Megasphaera  ↑  Atopobium  ↑  Gardnerella  ↑  Prevotella  ↑  Roseburia  ↑  Lactate↓ SCFAs↑ TMA↑ Amino acids↓ Biogenic amines↑ Glucose↑ Metabolite changes in CT, VVC and BV are associated with microbial-related metabolic activities. High levels of glucose not only enhance the nutritional substrate for Candida , but also increase Candida adhesion. Vaginal swabs 16S rRNA 81 Cervical dysplasia Lactobacillus  ↓  Fannyhessea  ↓  Megasphaera  ↓  Streptococcus  ↑  Escherichia  ↑  Staphylococcus  ↑  Bacillus  ↑  Fenollaria  ↑  Corynebacterium  ↑  Peptoniphilus  ↑  Acinetobacter  ↑  Inositol phosphate↑ Oxidative phosphorylation↑ Terpenoids and steroids↑ Hormone biosynthesis↑ Escherichia , Staphylococcus , and Bacillus , which were increased with the degrees of cervical lesions and cancer, showed a positive correlation with the up-regulated metabolic pathways. Vaginal secretion 16S rRNA sequencing of V3-V4 region 43 Crvical dysplasia HPV Cervical cancer Lactobacillus ↓ Prevotella ↑ Sneathia ↑ Atopobium ↑ Streptococcus ↑ Gardnerella ↑ Dipeptides↓ Amino acid↓ Nucleotide↑ Amino acid degradation↑ Atopobium contributed to the metabolism of amino acid (L-threonine, L-lysine), amino acid product (Glutathione disulfide), methylmalonate and succinate. Gardnerella was the main contributor to the hippurate metabolism. NAD + flux was mainly explained by the presence of Sneathia Cervicovaginal lavages and vaginal swabs 16S rRNA 44 Pregnancy BV-associated bacteria↓ Lactobacillus microbiome (first to second trimester)↑ Lactate↑ 4-hydroxyphenylacetate↑ Amino acids↑ SCFAs (Propionate and Acetate)↓ Biogenic amines↓ The reduction in the presence of diverse anaerobic bacteria lead to lower levels of SCFAs (e.g. propionate and acetate) and biogenic amines. Higher levels of valine, leucine and isolecine are associated with increased abundance of Lactobacillus . 4-hydroxyphenylacetate is an important product of metabolic activity in the healthy vaginal microbiome. Vaginal swabs at two times during the first trimester and the second trimester NAATs and Gram stain scoring system (Nugent score) 175 Pregnancy Lactobacillus  ↑ , BV-related genera (e.g., Prevotella , Atopobium , Sneathia )↓ during pregnancy Lactobacillus  ↑ , Gardnerella  ↑ , Prevotella  ↑ , Atopobium  ↑ , and Streptococcus ↑ at the puerperium Lactate↑ Sarcosine↑ Amino acids↑ Lactobacillus strongly positively correlated to lactate, many amino acids and sarcosine. BV-associated genera were positively correlated to putrescine, methylamine, tyramine, formate, TMA, alcohols (i.e., ethanol, isopropanol), and SCFAs (i.e., acetate, butyrate, propionate). Bifidobacterium , Streptococcus and Alloscardovia correlated with nucleotides, glucose, choline, benzoate, and fumarate Vaginal swabs at 9-13 weeks, 20-24 weeks and 32-34 weeks of gestational age, and puerperium (40-55 days after delivery) 16S rRNA sequencing of V3-V4 region 161 Preterm birth The vaginal microbiome clusters to well-defined CSTs Tyramine↑ Choline↑ Association between Dialister species or Enterococcus faecalis and tyramine. Cervicovaginal Samples between 20 and 24 weeks of gestation 16S rRNA sequencing of V3-V4 region 176 Preterm birth Unknown GalNAc↓ Sucrose↓ Steroid hormone↓ Proliferating pathogenic bacteria consume large amounts of sugar sources, resulting in an inadequate supply of GalNAc and sucrose for healthy Lactobacillus spp .. Disrupted steroid hormone biosynthesis pathway can facilitate pathogenic bacteria proliferation and weaken vaginal microecological immune system ability to clear pathogenic bacteria. Vaginal swabs at the posterior fornix between 31 and 36 gestational weeks Unknown 177 RIF Aerobic bacteria ( Escherichia , Enterococcus , Streptococcus , and Corynebacterium )↑ Anaerobic bacteria↑ 2’,3-cyclic UMP↑ Inositol phosphate↑ Fatty alcohol↓ Benzopyran↓ Glycerophospholipid↓ Naphthopyran↓ Benzopyran and glycerophospholipid were significantly positively correlated with the abundance of Lactobacillus . The decrease in glycerophospholipids contributed to a significant increase in inositol phosphates, one of the metabolites of glycerophospholipids, which affected embryo implantation. Upper third of the vagina on the day of embryo transfer before the operation 16S rRNA 22 Adverse embryo transfer Sphingobium  ↑  Corynebacterium  ↑  Ralstonia  ↑  Enterobacter  ↑  Enterococcus  ↑  Gardnerella ↑in EF and CL Pyrimidine metabolism (implantation success)↑ Biosynthesis of lysine metagenomes (implantation failure)↑ Biogenic amines↑ The L-lysine–BAs– Lactobacillus axis with the enhanced lysine biosynthesis indirectly resulting to changes in vaginal pH through BA production, thereby inhibiting Lactobacillus , stimulating the growth of other bacteria and destabilizing reproductive health. Pyrimidine nucleosides are life forms composed of DNA and RNA that play an important role in embryonic development. CL, CU, CV and EF 16S rRNA gene sequencing and metagenomic sequencing 101 Oocyte quality and growth Unknown Fatty acids↑ Vitamin B3↑ Vitamin D↑ Hormone↑ Fatty acids, vitamin and hormone pathways were associated with peak estradiol. Fatty acids appear to be important in the response to ovarian stimulation and potentially oocyte development. Vitamin B3 and vitamin D act as an antioxidant protecting the developing follicle from reactive oxygen species. Estrogen and C21 steroid hormone metabolism were associated with the number of mature oocytes. Follicular fluid Unknown 107 Infertility Unknown PUFAs↓ Progestin steroids↓ Carnitine↓ Acylcarnitines↑ Xanthine metabolites↑ PUFAs participate in female fertility at different reproductive phases, including oocyte maturation and quality, and embryo implantation. Progestin steroids contribute to fertility and the maintenance of pregnancy. Carnitine levels were correlated with the cytokine and cellular profile of endometriosis. High levels of acylcarnitines were associated with beta-oxidation dysfunction, participating in inflammation processes. Endometrial tissue sample Unknown 108 ESPL Unknown Ratio of n-6 PUFA/n-3 PUFA↑ Eicosanoids synthesis↓ Essential fatty acids (C18:3n-3 and C18:2n-6) ↓  Lysophosphatide↓ Diglyceride↓ The higher n-6 PUFA/n-3 PUFA ratio have inhibited the production of C20:5n-3-derived eicosanoids, thereby increasing the level of inflammation and ultimately promoting ESPL. The lower content of C18:3n-3 and C18:2n-6 in placenta of cases may lead to insufficient LCPUFA synthesis in embryo and hinder the normal embryonic development. Reducing lysophospholipid levels affects ovarian and placental function, endometrial tolerance and implantation by limiting LPA synthesis. Decidual and villous tissues Unknown 109 RPL Unknown Fatty acids↓ Sphingolipids↓ Carnitine↓ Glycerophospholipid↓ Sphingolipid metabolism and signaling pathways are closely related to trophoblast differentiation. Down-regulation of carnitine is associated with abnormal fatty acid β-oxidation in recurrent pregnancy loss. Decidual samples Unknown 110 VVC vulvovaginal candidiasis, BV bacterial vaginosis, DDP decaprenyl diphosphate, qPCR Quantitative polymerase chain reaction, SCFAs short-chain fatty acids, TMA trimethylamine, GalNAc N-acetyl-D-galactosamine, NAATs nucleic acid amplification techniques, RIF recurrent implantation failure, CL the lower third of the vagina, CU the posterior fornix, CV cervical mucus drawn from the cervical canal, EF endometrial fluid, PUFAs polyunsaturated fatty acids, LCPUFA long-chain polyunsaturated fatty acid, LPA lysophosphatide acid, ESPL early spontaneous pregnancy loss, RPL recurrent pregnancy loss Overview of the microbial and metabolic profiles of reproductive tract related to women’s physiological and reproductive health L. gasseri  ↓ L. crispatus  ↓ Streptococcus sp .↑ L. iners  ↑ Lactate↑ Acetate↑ Atopobium  ↑ Prevotella  ↑ Other anaerobes↑ L. iners  ↓ Lactate↓ Acetate↑ Succinate↑ Amino acids Sugars L. iners  ↑ Gardnerella  ↑ L-glutamate↑ DDP↑ Glycogen-related metabolites↑ P. bivia was positively correlated with DDP. L. iners was positively correlated with L-glutamate. These metabolites promoted peptidoglycan synthesis and led to Candida infection. Linoleic acid↑ Arachidonic acid↑ Phenylalanine↑ Tyrosine↑ Tryptophan↑ Sugar alcohols↑ Linoleic acid act as a pro-inflammatory factor in the response of the vaginal mucosa to C. albicans . L-phenylalanine may contribute to the development of VVC. Prevotella  ↑ Atopobium  ↑ Mycoplasma hominis  ↑ Amines↑ SCFAs↑ Nicotinate↑ Organic acids (Malonate and Acetate)↑ Bacterial participation in amino acid decarboxylation reactions to produce biogenic amines. SCFAs may be involved in the recruitment and activation of the innate immune cells in the female genital tract. Acetate was produced by Prevotella and Mobiluncus spp . Megasphaera sp. type 1  ↑ G. vaginalis  ↑ Eggerthella sp. type 1  ↑ Leptotrichia  ↑ Sneathia spp .↑ Prevotella timonensis  ↑ N-acetylneuraminate↑ 12-HETE↑ Arginine↓ Amines↑ N -acetylputrescine↑ Succinate↑ G. vaginalis can transport and catabolise N-acetylneuraminic acid as a carbon and energy source. BV-associated bacteria convert arachidonic acid to 12-HETE, a biomarker of inflammation. Increased utilization of amino acids by bacteria, as evidenced by high levels of amines and degradation products of amines. Lactobacillus ↓ G. vaginalis ↑ Atopobium ↑ Prevotella ↑ Veillonella ↑ M. hominis ↑ SCFAs↑ Organic acids (Succinate, Formate, Fumarate)↑ Biogenic amines↑ Hypoxanthine and xanthine↑ L. iners  ↑ Gardnerella  ↑ P. timonensis  ↑ Fannyhessea vaginae  ↑ Histidine↓ Arginine↓ Valine↑ Purine↓ Nicotinate and Nicotinamide↓ Eicosenoic acid↑ Carnitine↑ BV-associated bacteria tend to utilize amino acids as a carbon source. Strong association of valine upregulation with biofilm-forming bacterial species such as Prevotella and Sneathia . Eicosenoic acid, a naturally occurring monounsaturated fatty acid and an immune system stimulator, was found to be highly discriminatory for BV. BV VVC VVC_BV Candida (in VVC_BV and VVC)↑ G. vaginalis and Prevotella (in VVC_BV and BV)↑ Indole↑ Chorismate↑ Aromatic amino acid↑ Coenzyme A biosynthesis pathway related to fatty acid synthesis↑ Tryptophan is a known aromatic amino acid present in bacteria. Prevotella , Chlamydia , and certain bacteria associated with BV possess the ability to degrade tryptophan into indole. Fatty acids in the vagina could trigger elevated levels of inflammatory cytokines in VVC_BV. C. trachomatis VVC BV Faecalibacterium  ↑ Megasphaera  ↑ Atopobium  ↑ Gardnerella  ↑ Prevotella  ↑ Roseburia  ↑ Lactate↓ SCFAs↑ TMA↑ Amino acids↓ Biogenic amines↑ Glucose↑ Metabolite changes in CT, VVC and BV are associated with microbial-related metabolic activities. High levels of glucose not only enhance the nutritional substrate for Candida , but also increase Candida adhesion. Lactobacillus  ↓ Fannyhessea  ↓ Megasphaera  ↓ Streptococcus  ↑ Escherichia  ↑ Staphylococcus  ↑ Bacillus  ↑ Fenollaria  ↑ Corynebacterium  ↑ Peptoniphilus  ↑ Acinetobacter  ↑ Inositol phosphate↑ Oxidative phosphorylation↑ Terpenoids and steroids↑ Hormone biosynthesis↑ Crvical dysplasia HPV Cervical cancer Lactobacillus ↓ Prevotella ↑ Sneathia ↑ Atopobium ↑ Streptococcus ↑ Gardnerella ↑ Dipeptides↓ Amino acid↓ Nucleotide↑ Amino acid degradation↑ Atopobium contributed to the metabolism of amino acid (L-threonine, L-lysine), amino acid product (Glutathione disulfide), methylmalonate and succinate. Gardnerella was the main contributor to the hippurate metabolism. NAD + flux was mainly explained by the presence of Sneathia BV-associated bacteria↓ Lactobacillus microbiome (first to second trimester)↑ Lactate↑ 4-hydroxyphenylacetate↑ Amino acids↑ SCFAs (Propionate and Acetate)↓ Biogenic amines↓ The reduction in the presence of diverse anaerobic bacteria lead to lower levels of SCFAs (e.g. propionate and acetate) and biogenic amines. Higher levels of valine, leucine and isolecine are associated with increased abundance of Lactobacillus . 4-hydroxyphenylacetate is an important product of metabolic activity in the healthy vaginal microbiome. Lactobacillus  ↑ , BV-related genera (e.g., Prevotella , Atopobium , Sneathia )↓ during pregnancy Lactobacillus  ↑ , Gardnerella  ↑ , Prevotella  ↑ , Atopobium  ↑ , and Streptococcus ↑ at the puerperium Lactate↑ Sarcosine↑ Amino acids↑ Lactobacillus strongly positively correlated to lactate, many amino acids and sarcosine. BV-associated genera were positively correlated to putrescine, methylamine, tyramine, formate, TMA, alcohols (i.e., ethanol, isopropanol), and SCFAs (i.e., acetate, butyrate, propionate). Bifidobacterium , Streptococcus and Alloscardovia correlated with nucleotides, glucose, choline, benzoate, and fumarate Tyramine↑ Choline↑ GalNAc↓ Sucrose↓ Steroid hormone↓ Proliferating pathogenic bacteria consume large amounts of sugar sources, resulting in an inadequate supply of GalNAc and sucrose for healthy Lactobacillus spp .. Disrupted steroid hormone biosynthesis pathway can facilitate pathogenic bacteria proliferation and weaken vaginal microecological immune system ability to clear pathogenic bacteria. Aerobic bacteria ( Escherichia , Enterococcus , Streptococcus , and Corynebacterium )↑ Anaerobic bacteria↑ 2’,3-cyclic UMP↑ Inositol phosphate↑ Fatty alcohol↓ Benzopyran↓ Glycerophospholipid↓ Naphthopyran↓ Benzopyran and glycerophospholipid were significantly positively correlated with the abundance of Lactobacillus . The decrease in glycerophospholipids contributed to a significant increase in inositol phosphates, one of the metabolites of glycerophospholipids, which affected embryo implantation. Sphingobium  ↑ Corynebacterium  ↑ Ralstonia  ↑ Enterobacter  ↑ Enterococcus  ↑ Gardnerella ↑in EF and CL Pyrimidine metabolism (implantation success)↑ Biosynthesis of lysine metagenomes (implantation failure)↑ Biogenic amines↑ The L-lysine–BAs– Lactobacillus axis with the enhanced lysine biosynthesis indirectly resulting to changes in vaginal pH through BA production, thereby inhibiting Lactobacillus , stimulating the growth of other bacteria and destabilizing reproductive health. Pyrimidine nucleosides are life forms composed of DNA and RNA that play an important role in embryonic development. Fatty acids↑ Vitamin B3↑ Vitamin D↑ Hormone↑ Fatty acids, vitamin and hormone pathways were associated with peak estradiol. Fatty acids appear to be important in the response to ovarian stimulation and potentially oocyte development. Vitamin B3 and vitamin D act as an antioxidant protecting the developing follicle from reactive oxygen species. Estrogen and C21 steroid hormone metabolism were associated with the number of mature oocytes. PUFAs↓ Progestin steroids↓ Carnitine↓ Acylcarnitines↑ Xanthine metabolites↑ PUFAs participate in female fertility at different reproductive phases, including oocyte maturation and quality, and embryo implantation. Progestin steroids contribute to fertility and the maintenance of pregnancy. Carnitine levels were correlated with the cytokine and cellular profile of endometriosis. High levels of acylcarnitines were associated with beta-oxidation dysfunction, participating in inflammation processes. Ratio of n-6 PUFA/n-3 PUFA↑ Eicosanoids synthesis↓ Essential fatty acids (C18:3n-3 and C18:2n-6) ↓ Lysophosphatide↓ Diglyceride↓ The higher n-6 PUFA/n-3 PUFA ratio have inhibited the production of C20:5n-3-derived eicosanoids, thereby increasing the level of inflammation and ultimately promoting ESPL. The lower content of C18:3n-3 and C18:2n-6 in placenta of cases may lead to insufficient LCPUFA synthesis in embryo and hinder the normal embryonic development. Reducing lysophospholipid levels affects ovarian and placental function, endometrial tolerance and implantation by limiting LPA synthesis. Fatty acids↓ Sphingolipids↓ Carnitine↓ Glycerophospholipid↓ Sphingolipid metabolism and signaling pathways are closely related to trophoblast differentiation. Down-regulation of carnitine is associated with abnormal fatty acid β-oxidation in recurrent pregnancy loss. VVC vulvovaginal candidiasis, BV bacterial vaginosis, DDP decaprenyl diphosphate, qPCR Quantitative polymerase chain reaction, SCFAs short-chain fatty acids, TMA trimethylamine, GalNAc N-acetyl-D-galactosamine, NAATs nucleic acid amplification techniques, RIF recurrent implantation failure, CL the lower third of the vagina, CU the posterior fornix, CV cervical mucus drawn from the cervical canal, EF endometrial fluid, PUFAs polyunsaturated fatty acids, LCPUFA long-chain polyunsaturated fatty acid, LPA lysophosphatide acid, ESPL early spontaneous pregnancy loss, RPL recurrent pregnancy loss Despite being a low-biomass environment, the uterine cavity exhibits relatively high microbial diversity in both healthy and diseased states 84 , 85 . In healthy women, Lactobacillus is the most commonly reported dominant genus in the uterine microbiome 77 , 86 , although small amounts of other genera such as Prevotella , Gardnerella , Bifidobacterium , Atopobium , and Sneathia can also be detected 16 , 87 . However, some studies have shown that even in the absence of Lactobacillus , asymptomatic healthy women may have uterine microbiota dominated by genera such as Pseudomonas , Acinetobacter , Vagococcus , and Sphingobium 14 , suggesting that non- Lactobacillus -dominated microbiota may also be considered normal in the absence of pathological signs. The heterogeneity of the uterine microbiota is likely shaped by a combination of host and environmental factors, including genetic background, hormonal fluctuations, immune characteristics, and sexual intercourse 88 – 90 . Given its low-biomass nature, sampling procedures (such as transcervical or transvaginal collection), reagent contamination, and laboratory environments easily introduce exogenous microbes 91 , 92 . The lack of adequate negative and blank controls in current studies increases the risk of false positives or incorrect community structure inferences 93 – 95 . Moreover, differences in sample types (endometrial tissue versus uterine fluid) and sequencing approaches (targeting different 16S rRNA hypervariable regions or using shotgun metagenomics) result in significant variations in detected microbial profiles 96 – 98 . Defining a single “healthy” or “core” uterine microbiome in non-pregnant women, analogous to the vaginal microbiota, remains challenging based solely on microbial DNA (Fig. 1c ). However, this microbial heterogeneity may have different impacts on successful conception. The dominance of non- Lactobacillus species within the endometrial microbiota has been associated with reduced endometrial receptivity and an increased risk of recurrent implantation failure (RIF) 99 – 101 . An increased abundance of non- Lactobacillus bacteria (e.g., Atopobium , Neisseria , G. vaginalis , Ureaplasma , and Proteobacteria) has also been consistently observed in various types of miscarriage cases 102 – 106 . Currently, a Lactobacillus -dominated endometrial microbiome is considered an effective biomarker for predicting reproductive success 89 . Most current studies rely on 16S rRNA-based relative abundance data to describe correlations, with some reporting functional evidence from metabolic products in the uterine that may influence fertility 107 – 110 . Nevertheless, many questions remain regarding how the composition of the uterine microbiome, in both pregnant and non-pregnant states, influences functional dynamics and metabolism. Addressing these gaps is essential for elucidating the biological mechanisms through which the endometrial microbiota impacts female reproductive health. Similarly, the ovaries, which produce follicles, and the fallopian tubes, responsible for transporting oocytes, are not always sterile. At the opening of the fallopian tube, the proportion of microbes in the uterine cavity continues to increase, with the median relative abundance of Lactobacillus being only 1.69% (Fig. 1c ) 14 . This finding contrasts sharply with the Firmicutes-dominated microbiota (mainly Lactobacillus ) in the vagina. Most studies on the fallopian tube microbiome have reported the presence of various bacteria adapted to a weakly alkaline environment. The main taxa included Shigella , Bacteroides , Staphylococcus , Enterococcus , Corynebacterium , and Pseudomonas 15 , 89 , 111 , 112 , among which Shigella and Bacteroides are the most frequently reported representative taxa in cohorts of patients undergoing hysterectomy for benign gynecological conditions 15 . Enrichment of microbial diversity in the fallopian tubes has been linked to the progression of various gynecological conditions, notably salpingitis, infertility, and ectopic pregnancy 112 , 113 , suggesting a potential role of the upper genital tract (UGT) microbiota in maintaining tubal function. It is noteworthy that current study samples are mainly derived from patients who have undergone salpingectomy, hysterectomy for endometrial cancer, or hysterectomy for benign uterine and cervical diseases, which may limit the generalizability of findings on the fallopian tube microbiome. Future research should focus on developing non-invasive or minimally invasive sampling methods to improve sample diversity and conduct large multicenter cohort studies to enhance result generalizability. Studies on the ovarian microbiome have hardly been described, mainly focusing on reports related to malignant tumors 114 – 116 . The microbial composition of ovarian cancer tissues differs significantly from tissues from normal distal fallopian tubes, characterized by an increased ratio of Proteobacteria to Firmicutes 115 , 117 . Notably, Acinetobacter spp ., belonging to Proteobacteria, are widely and significantly enriched in ovarian cancer tissues 89 . Pediococcus , Staphylococcus , Sphingomonas , and Enterococcus have also been detected within tumor tissues 117 . These findings suggest that the altered abundance of certain bacteria in the ovarian microbiome may serve as potential biomarkers for ovarian cancer. Importantly, the increase in Acinetobacter spp. correlates with the host’s inflammatory gene expression profile 116 , indicating that multiple bacteria present in ovarian cancer tissues may be involved in inflammatory processes. However, a clear causal relationship between the microbiome and ovarian cancer has yet to be established. Moreover, infections by pathogens such as HPV, cytomegalovirus, Brucella , Mycoplasma , and Chlamydia trachomatis have been reported to be associated with ovarian cancer development, suggesting that infectious agents may play a role in tumorigenesis. Nevertheless, there is currently insufficient evidence to confirm the existence of a distinct and stable ovarian microbiome that can be reliably distinguished from contaminants 118 . Therefore, further comprehensive and methodologically rigorous studies are needed to elucidate the composition, functional roles, and clinical significance of the ovarian microbiome in both health and disease.

Numerous studies have shown the relationship between the vaginal microbiome and health outcomes. Dysbiosis greatly increases susceptibility to reproductive tract infections, such as human immunodeficiency virus (HIV) infection, herpes simplex virus type 2 infection, HPV, and adverse pregnancy outcomes 59 , 116 , 119 – 123 . In some cases, microbial transfer from the LGT to proximal sites of the reproductive system was frequently observed, indicating that cross-site microbial exchange may cause or aggravate a variety of reproductive diseases. The low-intensity inflammation and immune reactions shown in UGT infections are usually the result of bacterial action. G. vaginalis , A. vaginae , Sneathia amnionii , Prevotella , and Clostridium were found to be significantly enriched in the uterine cavity of UGT infections 124 – 126 . Notably, these bacteria were frequently detected in women with vaginal dysbiosis, suggesting a potential microbial linkage between the vagina and uterus 127 . Such cross-site microbial associations are manifested not only in the overlapping occurrence of specific taxa but also in coordinated alterations in community composition. For instance, women with a history of abortion or multiple vaginal deliveries exhibit increased similarity between their vaginal and uterine microbiomes 126 , likely reflecting alterations in the uterine microenvironment. The uterine microbiota of rats receiving vaginal microbiota transplants from patients with chronic endometritis (CE) also undergone marked structural changes 126 . These observations collectively support the potential for microbial translocation from the vaginal to the uterine niche. Furthermore, vaginal microbiota dysbiosis may propagate into the uterine cavity, thereby perturbing the uterine microenvironment and eliciting inflammatory responses 126 , 128 , 129 (Fig. 1d ). CST IV, which is predominantly associated with pathological states of the vaginal microbiota 130 , 131 , has a negative impact on the uterine microenvironment. This CST disrupts the phosphorylation state and integrity of tight junction complexes, thereby increasing the paracellular permeability of the cervicovaginal epithelium 132 . Such disruption may lead to cervical laxity or insufficiency, facilitating microbial exchange between the vaginal and uterine 133 . These evidences strongly highlight the dynamic interplay between the two microbial niches, highlighting the high mobility of certain vaginal bacteria and their potential to translocate between the LGT and the UGT 126 , 134 . This also reveals a critical interdependence between vaginal bacterial translocation, uterine microecology, and endometrial health. In the context of microbial translocation, many CST IV-associated bacteria are potent inducers of the inflammatory response. Megasphaera elsdenii and Prevotella timonensis promote the maturation of dendritic cells (DCs), leading to increased secretion of pro-inflammatory cytokines such as IL-1β, IL-6, IL-8, IL-12, and TNF-α 135 . These cytokines not only drive local inflammation but can also ascend from the upper vagina to the uterus, thereby influencing the uterine immune microenvironment and T cell differentiation 135 , 136 . (Fig. 1d ). This alteration in the immune milieu plays a crucial role in shaping adaptive immunity within the endometrium. Once activated, adaptive immune responses can lead to chronic inflammation and tissue damage, as is characteristic in pelvic inflammatory disease (PID) 137 (Fig. 1d ). In particular, T helper 1 (Th1) to T helper 2 (Th2) -mediated immune excursions may lead to fibrinous inflammatory damage along the epithelial surface of the fallopian tube, the peritoneal surface of the fallopian tube and the ovary 138 . The presence of certain bacterial species, such as Staphylococcus and Streptococcus , has also been correlated with the degree of fibrosis and severity of tissue injury 139 , suggesting a role in exacerbating pathological outcomes and further increasing the risk of adverse reproductive outcomes 137 , 138 . Importantly, these CST IV-associated microorganisms enhance the synergistic symbiotic effects of microbiota across sites and exacerbate clinical progression, such as increasing susceptibility to C. trachomatis and N. gonorrhoeae , key pathogens of PID, by 3.4-fold and 4.1-fold, respectively 53 , 140 , 141 . Existing studies have shed light on the relationship between microbial translocation and reproductive tract infections. On the downside, many studies have small sample sizes and are biased towards specific populations (e.g., Europe and the United States), with insufficient data on global diversity 12 , 60 , 142 – 145 . On a temporal scale, studies focused mostly on cross-sectional or transient effects but have lacked tracking of the long-term consequences of microbial translocations 131 , 146 – 148 . Sample size should be expanded to include women of different races, ages, geographic regions and lifestyles to improve the generalizability of the findings. Also, long-term follow-up studies are important to assess the potential impact of microbial translocation on women’s reproductive health, pregnancy outcomes and chronic diseases. Decreased fertility is influenced by age, unhealthy lifestyle factors such as poor diet, smoking, and environmental exposures 149 – 154 . Factors known to affect female fertility can also affect their microbiome. The role of the reproductive microbiome in participating in the process of female conception has been further emphasized with the strengthening of research on its regulation of the reproduction-related immune system. The dominance of Lactobacillus spp . in the reproductive tract of women of reproductive age is closely associated with higher rates of pregnancy, implantation, ongoing pregnancy, and live birth 16 , 103 , 155 , 156 . Particularly in early pregnancy, a shift toward Lactobacillus dominance is more pronounced in certain populations, such as women of African ancestry 157 – 159 . This change is closely related to the increased endogenous estrogen levels during pregnancy, indicating a positive correlation between estrogen and Lactobacillus abundance 26 . Sustained high estrogen levels help stabilize the microbial community structure, maintaining the stability and function of the reproductive tract environment, which may explain the relative stability of the vaginal microbiota during pregnancy 26 . Within this stable microenvironment, an optimal abundance of L. crispatus (~80%) is considered more favorable for successful conception, suggesting that reproductive success depends not only on the bacterial species but also on achieving an optimal microbial abundance 160 (Fig. 2 ). Conversely, non- Lactobacillus bacteria from UGT or LGT has been linked to increased risks of implantation failure, spontaneous abortion, and preterm birth and other pregnancy complications 1 , 85 , 157 , 161 – 168 . For example, abundance changes of BV-associated species such as G. vaginalis , A. vaginae , Proteus , Group B streptococcus and Prevotella bivia have been highlighted in cases of implantation failure, idiopathic infertility, miscarriage and preterm birth 161 , 169 – 174 . Importantly, the stability of vaginal bacteria in early pregnancy of women with miscarriage is similar to that of healthy surviving pregnancies with full-term delivery, indicating that once established, microbial patterns tend to persist and continue to influence reproductive outcomes 166 . Fig. 2 Factors that are crucial for successful conception and a healthy pregnancy. These factors are divided into three aspects: microbiome, metabolites, and immune regulation. The uterine cavity and proximal vagina often show moderate abundance of Lactobacillus and low biodiversity, which is conducive to successful conception and pregnancy 160 . These microbial changes are often accompanied by fluctuations in the metabolite levels of the surrounding environment 22 , 101 , 107 – 110 , 161 , 175 – 177 . Both are directly or indirectly involved in the immune regulation of female conception and throughout pregnancy, including stimulating natural killer (NK) cells, responding to antigen-presenting cells (APCs), and T cell differentiation. These play an indispensable role in maintaining the complex immune response at the maternal-fetal interface. The figure was created with BioRender.com. These factors are divided into three aspects: microbiome, metabolites, and immune regulation. The uterine cavity and proximal vagina often show moderate abundance of Lactobacillus and low biodiversity, which is conducive to successful conception and pregnancy 160 . These microbial changes are often accompanied by fluctuations in the metabolite levels of the surrounding environment 22 , 101 , 107 – 110 , 161 , 175 – 177 . Both are directly or indirectly involved in the immune regulation of female conception and throughout pregnancy, including stimulating natural killer (NK) cells, responding to antigen-presenting cells (APCs), and T cell differentiation. These play an indispensable role in maintaining the complex immune response at the maternal-fetal interface. The figure was created with BioRender.com. These microbial changes are often accompanied by fluctuations in the metabolite levels of the surrounding environment 175 – 177 (Fig. 2 ). Altered vaginal metabolic profiles in RIF include elevated inositol phosphate levels and reduced concentrations of benzopyran and naphthopyran, the latter of which is positively associated with Lactobacillus abundance 22 , 178 . Excess inositol phosphates induce intracellular Ca²⁺ influx and stimulate uterine contractions, thereby interfering with embryo implantation 22 , 179 . Moreover, dysbiosis disrupts the immune environment of the endometrium through the modulation of inflammatory mediators, thereby affecting reproductive outcomes 59 , 180 . Such alterations in the inflammatory milieu are particularly evident in cases of recurrent miscarriage, where aberrant levels of cytokines like interferon (IFN)-γ and IL-6 have been observed 165 . These cytokine abnormalities impair the function of endometrial immune cells, thereby disrupting the balance between immune activation and tolerance at the maternal-fetal interface 165 , 168 . During embryo implantation, an appropriate balance between Th1 and Th2 cells is critical for successful embryo implantation 181 . However, inflammatory cytokines induced by microbial stimulation or activated APCs interfere with the proper differentiation of T cell subsets, thereby reducing endometrial immune receptivity and increasing the risk of implantation failure 138 , 182 – 185 (Fig. 2 ). These lines of evidence highlight that dynamic interactions between the reproductive tract microbiota and the uterine immune system play a pivotal role in maintaining immune homeostasis and ensuring favorable reproductive outcomes. However, most studies have only revealed correlations between the vaginal microbiota and reproductive outcomes, while causal evidence demonstrating how the microbiota affects fertility remains insufficiently explored. Given the involvement of the reproductive tract microbiota in modulating immune responses at the maternal-fetal interface, an exploration of the immune mechanisms behind it seems to be the next step in the program. Only by analyzing the root causes of pregnancy failure can we provide guidance for future clinical treatment.

The vital role of microbes in women’s physiological well-being was acknowledged over a century ago. The first discovery of Lactobacillus (later called Döderlein Bacillus ) in the vaginal secretions of pregnant women 1 , 2 , is regarded as the beginning of vaginal microecology research. Since then, several bacteria that may be pathogenic to the vagina have been discovered. Notably, the notorious Neisseria gonorrhoeae and Haemophilus vaginalis , both recognized as pathogenic bacteria causing reproductive tract infections, were discovered and isolated at that time 3 , 4 . Although these studies laid the groundwork for clarifying the relationship between microbes and female physiological health, researchers were keen to identify pathogens but generally overlooked the importance of native commensal bacteria 5 – 7 . In recent decades, the understanding of microorganisms relevant to women’s physiological and even reproductive health has gone far beyond the scope of pathogen detection. More attention has been devoted to the physiological regulatory functions of vaginal lactobacilli, symbiotic microbes and the entire microbial community in these studies 8 – 10 . Microbiome-based ecology is becoming a major theme in this line of research. This is inextricably linked to the foundational data provided by the Human Microbiome Project 11 , which presented the dominance of various lactobacilli in the vaginas of healthy women at scales of geography, time, ethnicity, and physiological state 12 , 13 . The knowledge not only shed light on the diversity of the vaginal microbes, but also emphasize the broader relationship between the microbiome and host health. For example, an imbalanced vaginal microbiota is strongly associated with the development of diseases including bacterial vaginosis (BV), cervical cancer, and even increases the risk of infertility and preterm delivery 14 – 17 . These findings reinforce our understanding of the local microbes within the reproductive tract and elucidate the role they play in transitions between women’s health and disease. Nowadays, the study of the female microbiome is at an exponential stage of development. New research goals have emerged to comprehensively reconstruct the relationship between symbiotic microbes and the physiological state of the host, and to explore targeted intervention strategies. The horizon has been expanded to the entire reproductive system (including the upper and lower genital tracts) and cross-body-site microbial regulation. In contrast, the effectiveness of distal microbial influences in maintaining and regulating the female microbiota at different times (non-pregnancy and pregnancy) is still in the stage of ongoing research and validation 18 – 21 . Knowledge of microbial modulation of female physiological and reproductive functions across temporal and spatial scales is accumulating at an unprecedented rate. Therefore, this review aims to synthesize current findings on the role of the microbiome in female physiology and reproductive health. To support this synthesis, we conducted an extensive literature search using PubMed, employing combinations of terms such as ‘vaginal OR uterine microbiome’, ‘female reproductive health AND microbiome’, ‘pregnancy’, ‘endometrial receptivity’, ‘reproductive microbiota AND gut microbiome’, ‘hypothalamic-pituitary-ovarian (HPO) axis’, ‘probiotics AND women’s health’ to identify studies relevant to female physiological and reproductive processes. Articles in languages other than English were excluded. We primarily included peer-reviewed studies published between 2000 and 2025, covering clinical research as well as preclinical animal model studies. Our focus centers on the local effects of reproductive tract microbiota, proximal cross-site microbial interactions, and the distal regulatory roles of the gut microbiome. By integrating recent advances in microbial composition, functional characterization, and metabolite mediation, we aim to elucidate the potential mechanisms through which these microbial communities influence female physiological processes and/or reproductive outcomes across diverse spatial and temporal contexts. In addition, we discuss emerging microbiome-based strategies aimed at maintaining or restoring female health, offering perspectives for future research and potential clinical translation.