As the entrance part of the female reproductive tract, the vagina harbors the highest bacterial biomass, and it has been confirmed that there is a resident microbiome in the vagina (Chen et al., 2017; Ravel et al., 2011; Integrative, 2019). At the genus level, there is a higher relative abundance of Lactobacillus (more than 89%), while the presence of Prevotella, Sneathia, Staphylococcus, Veillonella, Streptococcus and others is still controversial (Chen et al., 2017; Ravel et al., 2011; Santella et al., 2022). Some scholars believe that the dominance of Lactobacillus in the vaginal microbiome represents a healthy, normal microbial environment, and the relative abundance of the dominant Lactobacillus determines the type of bacterial community, which is called the community state types (CSTs) (Kroon et al., 2018; Lloyd-Price et al., 2017; Younes et al., 2018). The CSTs are divided into five distinct CSTs, namely, CST I (dominated by L. crispatus), CST II (dominated by L. gasseri), and CST III (dominated by L. iners), CST IV [polymicrobial microbiome including Lactobacillus and BV-associated bacteria (BVAB)], CST V (dominated by L. jensenii) (Lloyd-Price et al., 2017; France et al., 2020). Currently, CSTs I, III, and IV, which have been extensively studied, are common in women, but CSTs II and V are rarely found (Doyle et al., 2018). Studies have shown that a vaginal microbiome dominated by L. crispatus (CST I) always maintains vaginal health, whereas a vaginal microbiome dominated by L. iners (CST III) is more prone to vaginal diseases (Jakobsson and Forsum, 2007; Verstraelen et al., 2009). Specific microbial taxa in the vagina can affect vaginal health and diseases by regulating inflammatory factors and their metabolites, hence necessitating an in-depth investigation of their interrelationship (De Seta et al., 2019; Breshears et al., 2015; Fuochi et al., 2019).

Over the years, it has been widely believed that the cervical microbiome is a continuation of the vaginal microbiome; however, recent evidence has confirmed differences between the vaginal and cervical microbiome (Chen et al., 2017). It has been found that among the cervical microbiome, Firmicutes is considered to be the most abundant phylum, and Lactobacillus is the main genus in this phylum (as high as 80.2%) (Onywera et al., 2019a; Onywera et al., 2019b). Keburiya et al. demonstrated that L.crispatus in the cervix could produce lactic acid and antimicrobial compounds, inhibit inflammation, thereby reducing the incidence of human papilloma virus (HPV) infection (Keburiya et al., 2022). Bacteroidetes is the second most abundant phylum, of which Prevotella is the dominant genus. Prevotella, as an important member of the cervical microbiome, affects the development of cervical lesions and persistent HPV infection through host nuclear factor kappa B (NF-κB)/C-myc during HPV infection in women of reproductive age (Dong et al., 2022). The next most abundant phyla are Actinobacteria and Fusobacteria, with Gardnerella and Sneathia being the most abundant genera, respectively (Onywera et al., 2019a; Onywera et al., 2019b). Intriguingly, higher abundances of Gardnerella and Sneathia were found in high-risk HPV-infected women compared to low-risk HPV-infected or non-HPV-infected women, suggesting that they may be closely related to high-risk HPV infection (Onywera et al., 2019a). It is imperative to enhance our understanding of the composition of the cervical microbiome and explore it plays a pivotal role in the screening and diagnosis of cervical HPV infection.

Throughout the past century, the uterine cavity has traditionally been perceived as a sterile environment devoid of microbial colonization (Heinonen et al., 1985). However, accumulating evidence suggests a low abundance and high diversity of microbiome colonize the endometrium (Moreno et al., 2016; Chen P. et al., 2021; Franasiak et al., 2016; Verstraelen et al., 2016). Some studies showed that Lactobacillus, Sphingobium, Acinetobacter, Methylobacterium and Streptococcus dominated the endometrium (Chen P. et al., 2021). Other studies assumed that Lactobacillus and Flavobacterium could represent the majority of the endometrial microbiome (Franasiak et al., 2016). Moreno et al. reported that Lactobacillus (71.7%), Gardnerella (12.6%), Bifidobacterium (3.7%), Streptococcus (3.2%), and Prevotella (0.866%) were the most common bacteria in the endometrium (Moreno et al., 2016). There is no consensus on the core microbiome of the endometrium in healthy women, but Lactobacillus is a consistent discovery, and it is considered one of the endometrium-resident microbes (Moreno et al., 2016). Previous research revealed that the endometrial microbiome was dominated by Lactobacillus (LD, Lactobacillus>90%, other bacteria<10%), and the clinical pregnancy rate and live birth rate were higher, which might predict the reproductive success; when the relative abundance of Lactobacillus was low (Lactobacillus<90%, other bacteria>10%), identified as microbial dysbiosis, the incidences of adverse pregnancy outcomes such as recurrent spontaneous abortion, preterm birth, biochemical pregnancy, and recurrent implantation failure (RIF) were increased (Moreno et al., 2016). Currently, the endometrial microbiome is considered an effective biomarker for predicting reproductive success rate, which could provide new insights and research directions for the prevention and treatment of adverse pregnancy outcomes (Punzón-Jiménez and Labarta, 2021).

Compared to the lower reproductive tract and the uterus, there are relatively few studies on the tubal microbiome (Miles et al., 2017; Zhou et al., 2019; Walther-António et al., 2016; Pelzer et al., 2018a; Canha-Gouveia et al., 2023). Walther-António et al. studied the whole reproductive tract microbiome of 31 patients with total hysterectomy and bilateral adnexectomy. The results revealed that Shigella and Bacteroides were the most important taxa in the fallopian tube (Walther-António et al., 2016). A study of the tubal microbiome based on salpingectomy showed that the main taxa included Staphylococcus, Enterococcus, Corynebacterium and Lactobacillus (Pelzer et al., 2018a). On the whole, the fallopian tube contains a variety of bacteria suitable for growth in a weakly alkaline environment, and the proportion of Lactobacillus is extremely low, which is markedly different from the microbiological composition of the lower reproductive tract and endometrium (Chen et al., 2017; Punzón-Jiménez and Labarta, 2021; Peric et al., 2019). In most studies on the tubal microbiome, the selected subjects tended to have benign diseases of the uterus and cervix that may affect the cervical physiological barrier, making it easy for bacteria (such as Lactobacillus) that colonize the lower reproductive tract to migrate upward (Walther-António et al., 2016; Pelzer et al., 2018a). A recent study comparing the microbiome of the fallopian tube in women who underwent tubal ligation (normal controls) and those who underwent hysterectomy for benign disease (cases) showed no significant differences in microbial diversity or differential abundance analysis (Canha-Gouveia et al., 2023). It was also found that the most prevalent genera among fallopian tube samples were Lactobacillus, Prevotella, Acinetobacter, Propionibacterium, and Faecalibacterium (Canha-Gouveia et al., 2023). This study is a significant impetus for further investigation into the microbial communities within the fallopian tubes. In the future, the normal composition of the tubal microbiome should be further explored to enrich the study of upper genital tract microbiome composition and reach a consensus on the composition of the tubal microbiome as soon as possible.

Until now, studies on the ovarian microbiome have hardly been described, mainly focusing on patients with gynecological tumors, and the ovarian microbiome of normal individuals has been particularly rare (Miles et al., 2017; Zhou et al., 2019; Banerjee et al., 2017). In a recent study, Corynebacterium, Blautia, Escherichia, Lactobacillus, and Trabulsiella were found to be highly enriched, while Lactobacillus was significantly decreased in ovarian samples from patients with malignant disease (Miles et al., 2017). Compared to normal ovarian samples, ovarian cancer samples exhibited significant differences in the composition of their ovarian microbiome. Pediococcus was the most commonly detected microbe, followed by Acinetobacter, Staphylococcus, Sphingomonas, Enterococcus, Chryseobacterium, and Burkholderia (Banerjee et al., 2017). Furthermore, the microbiome in malignant ovarian tissue displayed distinct microbial signatures when compared to the healthy surrounding ovarian tissues within the same individuals. Specifically, potentially pathogenic intracellular microorganisms, such as Acinetobacter, Chlamydia and Mycoplasma, were detected in 60%~76% of ovarian cancer cases (Zhou et al., 2019; Banerjee et al., 2017). These studies indicated that the reduction or disappearance of Lactobacillus in the ovarian microbiome and the increase in certain bacteria in the ovarian microbiome could potentially serve as biosignatures for the presence of gynecological tumors. Similarly, normal ovarian tissue has been found to be colonized by microbes (Zhou et al., 2019; Banerjee et al., 2017). So, what are the normal microbial communities found in the ovaries? The answer remains elusive due to the challenges in obtaining normal ovarian samples.

The vagina is a muscular canal connecting the cervix to the external genitalia, with the anterior part of the vagina located near the bladder neck and urethra and the posterior part adjacent to the rectum and anus (O’Connell et al., 2008). The structure of the vagina can be divided into the vaginal epithelium (predominantly composed of multilayered stratified squamous epithelial cells), lamina propria, and fibromuscular layer from inside to outside (Mazloomdoost et al., 2017). The uppermost layer of the vaginal epithelium consists of flat cells that lack classical cell-cell adhesion, and this layer of cells undergoes rapid shedding and regeneration throughout the menstrual cycle (Anderson et al., 2014; Blaskewicz et al., 2011; Gorodeski et al., 2005; Patton et al., 2000). Furthermore, this layer of cells can use intracellular glycogen deposition to produce lactic acid through anaerobic metabolism under hypoxic conditions and secrete hydrogen ions and glycogen to the vaginal lumen through V-H⁺-ATPase (Gorodeski et al., 2005).

Vaginal mucosal immunity includes innate immunity and adaptive immunity, constituting a comprehensive defense mechanism (Bojang et al., 2021). Innate immune cells, such as vaginal epithelial cells (VECs), neutrophils (NEUs), dendritic cells (DCs), macrophages (Mφs), natural killer (NK) cells, and mast cells (MCs), express pattern recognition receptors (PRRs) on their surfaces, including Toll-like receptors (TLRs), C-type lectin receptors (CLRs), Nod-like receptors (NLRs), and RIG-I-like receptors (RLRs) (Bojang et al., 2021; Kalia et al., 2019). These PRRs interact with pathogen-associated molecular patterns (PAMPs) to activate circulating monocytes, NEUs, and Mφs in tissues (Bojang et al., 2021; Kalia et al., 2019). Among them, Mφs are commonly localized in the lamina propria of vaginal tissue, exhibiting dual functionality by secreting pro-inflammatory cytokines and modulating the formation of various inflammasome complexes (NLRP1 and NLRP3) under the action of various microbial stressors (Schroder and Tschopp, 2010). The NLRP3 inflammasome can be activated by various stimuli, including bacterial, viral, and mitochondrial damage, thereby leading to the development of bacterial vaginosis (Zhao et al., 2018; Xiang et al., 2021). Pro-inflammatory cytokines and antimicrobial peptides (AMPs) secreted by these innate immune cells, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, IL-8, lactoferrin, human defensin-5, secretory leukocyte protease inhibitor (SLPI), elastin, ductin, and human beta-defensin-1 and -2, may be concentrated within the intracellular and/or extracellular matrix (Blaskewicz et al., 2011; Patel et al., 2013; Cole and Cole, 2008). Moreover, loosely attached VECs can create a distinct microenvironment susceptible to infiltration and penetration by resident immune cells, pro-inflammatory cytokines, and AMPs (Anderson et al., 2014). VECs also release anti-inflammatory cytokines, such as IL-10 and TGF-β, which regulate the inflammatory response and prevent excessive local cell damage (Steele and Fidel, 2002). In contrast, adaptive immunity can elicit pathogen-specific defense mechanisms by processing antigens derived from the pathogen by antigen-presenting cells (APCs) and subsequent presentation to T cells, thereby inducing T-cell activation. Following antigen presentation, antibody synthesis is activated, and cytokine production ensues. At the same time, the presence of plasma cells secreting IgG and IgA in the vagina is comparatively limited compared to the lamina propria of the cervix (Wira et al., 2005; Moragues et al., 2003).

Various microbes with potential commensal, symbiotic or pathogenic relationships inhabit the vagina and interact with its anatomy, histology, and immunity, thereby affecting vaginal health (Maseroli and Vignozzi, 2020; Balakrishnan et al., 2022). Endogenous Lactobacillus can colonize the vaginal intraepithelial and epithelial cells, enabling them to obtain nutrients and energy from glycogen stored within the epithelial cells under a slightly acidic environment (Anderson et al., 2014; Gorodeski et al., 2005). Lactobacillus, in turn, produces lactic acid, thereby inducing further acidification of the vaginal cavity and creating an inhospitable environment for many disease-causing bacteria and viruses (Miller et al., 2016). For example, under acidic conditions, the viability of sexually transmitted pathogens such as BVAB, Chlamydia trachomatis, herpes simplex virus-2, and human immunodeficiency virus significantly decreases (O’Hanlon et al., 2011; Gong et al., 2014; Conti et al., 2009; Aldunate et al., 2013). Specifically, when the pH value is below 4.5, lactic acid can induce inactivation of various BVAB (O’Hanlon et al., 2011). If there is a sufficient amount of lactic acid to lower the vaginal pH to below 4, it can efficiently inhibit Chlamydia trachomatis infection (Gong et al., 2014). In addition, lactic acid was observed to attenuate the release of pro-inflammatory cytokines from epithelial cells (Vallor et al., 2001). In particular, lactic acid induced the release of the anti-inflammatory cytokine and IL-1 receptor antagonist in vitro and inhibited the TLR-mediated production of pro-inflammatory cytokines (Chopra et al., 2022). Therefore, lactic acid reduces excessive damage to epithelial cells, which is beneficial for maintaining the integrity of vaginal epithelium. L. crispatus can produce both L- and D-lactic acid, leading to an increase in the levels of D-lactic acid and the ratio of D- to L-lactic acid, which has an impact on preventing upper reproductive tract infections (Witkin et al., 2013). Moreover, its production of hydrogen peroxide (H₂O₂) can prevent colonization by anaerobes, and there is an iron transport system in the nuclear genome of L. crispatus, which may hinder vaginal pathogens from obtaining iron (France et al., 2016). The microbial community dominated by L. iners is unstable and can only produce L-lactic acid, which is prone to transition to CST-IV (France et al., 2020). These findings suggest that L. crispatus may be more effective in protecting hosts from pathogens than L. iners (France et al., 2020; Witkin et al., 2013; France et al., 2016).

Pathogenic bacteria are also constituents of the vaginal microbiome residing in the mucosal layer. Generally, the periodic shedding of vaginal epithelial cells facilitates the elimination of pathogenic bacteria adhering to them, thereby serving as a natural defense mechanism (Patel et al., 2013; Maseroli and Vignozzi, 2020; Godha et al., 2018). In turn, pathogenic bacteria can breach the vaginal epithelial barrier. For instance, BVAB such as Gardnerella and Prevotella can produce sialidase, which potentially contributes to mucin cleavage and damage to vaginal epithelial cells (Muzny et al., 2020). VECs release pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, which promote the migration of local immune cells to the lesion site and initiate an immune defense to eliminate pathogenic bacteria (Balakrishnan et al., 2022; Fernando et al., 2014). When a significant number of pathogenic bacteria, such as BVAB, aggregate in the vagina and induce vaginal dysbiosis, it could increase susceptibility to sexually transmitted diseases. Pathogenic bacteria that caused intense vaginal inflammation could not only lead to increased levels of IL-1β, IL-17, and IL-23, as well as high recruitment of CCR5⁺ CD4 T cells, but they might also be strongly associated with increased susceptibility to human immunodeficiency virus (Gosmann et al., 2017). Vaginal dysbiosis induced by antibiotic treatment relies on high levels of IL-33 to suppress the adaptive immune response mediated by T cells, thereby impeding antiviral immunity against herpes simplex virus-2 infection in the mucosa (Torcia, 2019). Collectively, the interactions between the vaginal microbiome and the epithelium, as well as vaginal immunity, intricately regulated its innate and adaptive immune mechanisms, thereby governing the homeostasis or dysbiosis of the vaginal microbiome.

The cervix is the gateway from the vagina to the uterus and is divided into the upper and lower parts by the top of the vagina (Ludmir and Sehdev, 2000). The upper part accounts for 2/3 of the entire cervix, and its epithelium is a single layer of tall columnar epithelium. The lower part of the cervix extends into the vagina and is covered by multilayered stratified squamous epithelium with a smooth surface (Balcacer et al., 2019). The junctions between epithelial cells include tight junctions, adherence junctions, and desmosome junctions, which form an epithelial barrier with cervical epithelial cells (Blaskewicz et al., 2011). The columnar epithelium primarily comprises tight junctions, while squamous epithelium mainly comprises adherence junctions and desmosome junctions (Wira et al., 2010). The cervical stroma is located beneath the epithelial cells and is separated from them by the basement membrane. The stroma primarily consists of an extracellular matrix supplemented by fibroblasts, immune cells, elastin, proteoglycans, and hyaluronan (Tantengco et al., 2021).

Cervical epithelial tissue is mainly composed of epithelial cells and T cells, with CD8⁺ T cells slightly more abundant than CD4⁺ T cells among the latter. The distribution of these cells is highest in the squamous and columnar transformation areas of the cervix, and lowest in the endocervix (Pudney et al., 2005). The cervix is an organ where cellular immunity predominates (Pudney et al., 2005). Once pathogenic bacteria invade, APCs recognize them through PRRs, such as TLRs and NLRs, and subsequently present the antigen to T cells, thereby initiating cellular immunity (Pudney et al., 2005). T cells are stimulated to differentiate into effector T cells, some of which develop into memory T cells. Memory T cells are divided into central memory T cells (TCMs), which are stored in the extralymphoid tissues, and effector memory T cells (TEMs), which can travel between the blood and the extralymphoid tissues and perform their functions (Gebhardt et al., 2013; Sathaliyawala et al., 2013). Some memory T cells undergo differentiation into tissue-resident memory T cells (TRMs). CD8⁺ TRMs (CD69⁺, CD103⁺) constitute the major subset of CD8⁺ T cells in cervical tissues and are also defined as inflammatory mucosal T cells (Tims). When TRMs recognized homologous peptides, they released the cytokines IFN-γ, TNF-α, and IL-2, thereby upregulating adhesion molecules and chemokines and promoting the recruitment of memory T cells and B cells to tissues (Rosato et al., 2017; Schenkel et al., 2014).

There is a symbiotic relationship between L. crispatus and the cervical mucosal epithelium; L. crispatus can secrete lactic acid to increase the expression of cervical epithelial barrier proteins claudin1 and claudin4 to some extent, and its supernatant can also alleviate the increase in miRNA expression induced by pathogenic bacteria (Delgado-Diaz et al., 2022; Anton et al., 2017; Anton et al., 2018). Conversely, the supernatants of pathogenic bacteria, including Mobiluncus mulieris and Gardnerella vaginalis, increased the permeability of cervical cells and the expression of miR-143 and miR-145, thereby reducing the proliferation of epithelial cells and promoting the breakdown of the cervical epithelial barrier (Anton et al., 2017; Anton et al., 2018). Researchers also have discovered that Prevotella bivia, Sneathia amnii, Fusobacterium gonidiaformans, and Fusobacterium nucleatum increase the levels of 2-hydroxyglutarate, while Eggertella and Mobiluncus decrease cysteinylglycine and cysteinylglycine disulfide levels (Łaniewski and Herbst-Kralovetz, 2021; Maarsingh et al., 2022; McKenzie et al., 2021). These findings suggested the potential contribution of cervical pathogens to necrosis and apoptosis of cervical epithelial cells through oxidative stress pathways. Additionally, cervical pathogens induced elevations of inflammatory factors such as IL-1β, macrophage inflammatory protein (MIP)-3α, and IL-8, which exhibited the strongest correlation with neutrophil proteinases [matrix metalloproteinase-9 (MMP-9) and MMP-8], suggesting potential damage to epithelial integrity (Mohammadi et al., 2022). In vitro culture models further confirmed that IL-1β triggered the p38 and c-Jun N-terminal kinase (JNK) signaling pathways, decreasing tight junctions and impairing epithelial integrity (Kobayashi et al., 2021). Hence, the interaction between the cervical epithelium and the microbiome can affect the function of the cervical epithelial barrier. Current research on the impact of cervical microbiome on cervical immunity primarily focuses on the recruitment and differentiation of DCs, Mφs, and T cells (Anton et al., 2022; Liu et al., 2021; Zariffard et al., 2005; van Teijlingen et al., 2020; Jan et al., 2012; Jang et al., 2017). Gardnerella vaginalis has been shown to activate TLR2/4 and induce an immune response in cervical cells while exerting minimal impact on DCs stimulation and promoting differentiation of M2 Mφs toward the M1 phenotype (Anton et al., 2022; Liu et al., 2021; Zariffard et al., 2005). Megasphaera elsdenii and Prevotella timonensis significantly enhanced DCs maturation and promoted T-cell differentiation towards the Th1 phenotype in the cervix (van Teijlingen et al., 2020). In contrast, cervical Lactobacillus inhibited the pro-inflammatory response of epithelial and immune cells, promoted the differentiation of CD4⁺ T cells into immunosuppressive regulatory T cells (Tregs), and did not affect DCs maturation (Jan et al., 2012; Jang et al., 2017). In general, the interactions of the microbiome with the cervical epithelium and immunity played crucial roles in maintaining both the epithelial integrity and immune barrier functionality within the cervix.

The cervix undergoes multiple physiological changes throughout different stages of pregnancy. Extensive remodeling of the cervix is required to allow a full-term fetus to pass through the birth canal, including softening, maturation, dilation during labor, and postnatal repair (Read et al., 2007). In the first and second trimesters, the cervix remains tightly closed to maintain pregnancy by keeping the fetus securely within the uterus (Barrios De Tomasi et al., 2019). As delivery approaches in the third trimester, the cervix gradually softens and matures, transitioning from a closed state to complete dilation, which enables smooth delivery of the fetus. After delivery, the cervix quickly returns to a tightly closed state (Read et al., 2007). Meanwhile, the physiological changes of cervical remodeling are accompanied by alterations in its stroma, such as enhanced collagen solubility and relaxation of collagen matrix (Read et al., 2007; Yellon, 2019). Gardnerella vaginalis, Atopobium vaginae, Prevotella bivia, and Pseudonocardia asaccharolytica could induce or secrete MMP-1, MMP-9, and MMP-10, while also exhibiting collagen (type I and IV) degradation abilities (Tantengco et al., 2021; Łaniewski and Herbst-Kralovetz, 2021; Lithgow et al., 2022; Short et al., 2021). An animal experiment demonstrated that the colonization of Gardnerella vaginalis in the reproductive tract of mice led to increased dispersion of collagen fibers, indicating accelerated cervical remodeling (Sierra et al., 2018). Conversely, L. crispatus did not exhibit any influence on MMP expression and type I collagen degradation (Tantengco et al., 2021; Łaniewski and Herbst-Kralovetz, 2021; Lithgow et al., 2022; Short et al., 2021). In addition, L. iners and L. crispatus can modulate the upregulation or downregulation of inflammatory bacterial signals, which may be closely associated with cervical remodeling during parturition (Anderson et al., 2014; Doerflinger et al., 2014). Taken together, symbiotic bacteria and pathogenic bacteria residing in the cervix may potentially alter the structural integrity of cervical tissue structure through induction or secretion of MMPs, collagen degradation, and inflammatory bacterial signals modulation, which affects cervical remodeling during pregnancy in humans.

The uterus, a symbolic organ within the female reproductive tract, has thick-walled muscles and a hollow cavity shaped like a slightly flattened, inverted pear. Anatomically, the uterus is connected to the outside environment through the vagina and is adjacent to the rectum and bladder (Zhu et al., 2022). The uterus comprises the endometrium, myometrium, and serosa, with the endometrium serving as the initial attachment site of the embryo (Yang et al., 2019). According to the physiological structure, the endometrium is divided into two layers—the upper functional and lower basal layers. During the menstrual cycle, the upper functional layer of the endometrium is shed from the lower basal layer and then regenerated, due to changes in estrogen and progesterone (Inversetti et al., 2023). The endometrium is composed of two distinct cell types: endometrial epithelial cells, characterized by their simple columnar morphology, and endometrial stromal cells. Endometrial stromal cells transform into specialized secretory decidual cells during pregnancy, and endometrial epithelial cells also promote this process by secreting various factors (Liu et al., 2022).

The endometrium distributes a large number of immune cells, primarily uterine natural killer (uNK) cells, along with a small proportion of B cells and CD8⁺ T cells, which can generate immune mediators (Zhu et al., 2022). Immune mediators in the endometrial fluid and surface act as a barrier against direct contact between pathogens and epithelial cells while exhibiting bactericidal activity against gram-negative and gram-positive bacteria (Gershon and Dekel, 2020; Jost et al., 2014; Romero et al., 2014). It is worth noting that the number of immune cells is closely related to hormone levels, and during early pregnancy, immune cells may be as high as 30%~40% of the total number of cells in the endometrium (Zhu et al., 2022). The most abundant decidual immune cells are uNK cells, which account for 70% of the total (Yang et al., 2019). There are also Tregs, NEUs, DCs, Mφs, and MCs (Yang et al., 2019). Maintaining a normal pregnancy relies on establishing and stabilizing the maternal-fetal immune tolerance of the endometrium (Yang et al., 2019). During pregnancy, Th1 cells undergo a phenotypic switch to Th2 cells, resulting in the downregulation of the expression of Th1 cytokines IL-2, IFN-γ and TNF-α; Th17 cells transform into Tregs, and the inhibitory cytokines IL-10 and TGF-β secreted by Tregs play an immunosuppressive role (Yang et al., 2019). These mechanisms enable the fetus to evade maternal rejection throughout pregnancy, and the NF-κB signaling pathway plays a central role in regulating the immune response (Yang et al., 2019).

The endometrium provides a suitable area for the colonization of tissue-resident microbiome and modulates the endometrial microbiome through diverse pathways, including hormonal regulation and mucosal barriers (Zhu et al., 2022; Inversetti et al., 2023). Fluctuations in steroid hormones, such as estrogen and progesterone, during the menstrual cycle, can affect the composition of the endometrial microbiome. In particular, there are significant differences in the composition of the endometrial microbiome during the proliferative and secretory phases (Zhu et al., 2022; Inversetti et al., 2023). In the secretory phase of the menstrual cycle, endometrial epithelial cells proliferate to form a layer of adenosine cells. These adenosine cells are closely linked to create a powerful anatomical barrier that prevents resident bacteria from being exposed to the uterine immune system, thereby regulating the endometrial microbiome (Habiba et al., 2021).

Similarly, the study by Sola-Leyva et al. indicated that there is a low-biomass active microbiome in the endometrium of healthy women, which produces metabolites such as prostaglandins and tryptophan that exhibit antibacterial and immunomodulatory activities, thereby affecting endometrium function (Sola-Leyva et al., 2021). Numerous studies have consistently found that a Lactobacillus-dominated endometrium has a higher rate of successful embryo implantation (Moreno et al., 2016; Iwami et al., 2023). In vitro experiments, Kim et al. demonstrated that protein-like moieties secreted by Lactobacillus rhamnosus GR-1 have a unique ability to inhibit the production of pro-inflammatory cytokines in human myometrial cells, suggesting that Lactobacillus may inhibit maternal immune response and facilitate implantation of embryos (Kim et al., 2019). In germ-free mice colonized by Bacteroides Fragilis, the signaling pathway with TLR2 was activated due to the secretion of polysaccharide A (PSA), thereby inducing differentiation of Th1 cells and establishing an appropriate Th1/Th2 balance (Mazmanian et al., 2005). Therefore, it can be speculated that Bacteroides may modulate Th1/Th2 balance and maternal-fetal immune tolerance through PSA during pregnancy (Mazmanian et al., 2005; Inversetti et al., 2023). A high bacterial biomass or the presence of certain bacteria in the endometrium, such as Fusobacterium and Jonquetella, may be related to immune overstimulation and tissue destruction (Pelzer et al., 2018b). When the endometrial microbiome is dysbiosis, pathogenic bacteria can trigger the relevant signaling pathway to induce the release of pro-inflammatory cytokines, thus promoting the imbalance in Th17/Tregs, resulting in the RIF of the embryo (Chen P. et al., 2021). Altogether, the reciprocal interactions among the endometrium, immunity, and endometrial microbiome exert an influence on the uterine mucosal barrier and embryo implantation, highlighting the need for further investigation into their intricate interrelationships.

The fallopian tubes are a pair of slender and curved muscle tubes divided into four parts: the stroma, the isthmus, the ampulla, and the infundibulum. Their medial side is connected to the uterine horn, while their external end is free and umbrella-like (Eddy and Pauerstein, 1980). The fallopian tube is similar to other hollow organs, as its wall consists of sequential layers of mucosa, muscular, and serosa from the inside to the outside (Li and Winuthayanon, 2017). The mucosal epithelium of the fallopian tube comprises a single layer of tall columnar cells, which can be classified into three types: ciliated cells, secretory cells, and peg cells (Rigby et al., 2022). Among these types, ciliated cells and secretory cells are the main ones. Ciliated cells are primarily situated in the fimbriae of the uterine tube, accounting for more than 50% of its distribution, and secretory cells are mainly located in the isthmus, comprising approximately 60% of the entire mucosal epithelium, and are chiefly responsible for secreting active ingredients into the fallopian tube fluid (Pérez-Cerezales et al., 2018).

Immune cells are the prominent participants in the immune response of the fallopian tube and can be divided into innate immune cells and adaptive immune cells. These cells are mainly distributed in the epithelium and lamina propria of the fallopian tube, playing an indispensable role in establishing pregnancy and eliminating pathogens (Lee et al., 2015). The innate immune cells typically include NEUs, DCs, NK cells, MCs, and Mφs, but the dominant cell population has not been determined (Lee et al., 2015). According to most studies, T cells are described as the main population of immune cells in healthy fallopian tubes among the adaptive immune cells, accounting for 40%~60% of all leukocytes (Lee et al., 2015). In contrast, B cells constitute a relatively small proportion (5%~10%) (Lee et al., 2015). The epithelial cells forming the epithelial barrier of the fallopian tubes express cell receptors to detect pathogens and transmit defense response signals. Besides, they can also secrete cytokines and AMPs to stimulate and regulate immune responses (McGlade et al., 2022; Schleimer et al., 2007).

The fallopian tube is the site where the ova and sperm combine and the channel for transporting the zygote (Li and Winuthayanon, 2017). The fluid environment secreted by the tubal secretory cells is rich in nutrients, such as proteins, adhesion molecules, specific glycoproteins, and inorganic salts, providing favorable conditions for microbial proliferation (Pérez-Cerezales et al., 2018). The mucosal epithelium of the fallopian tube has a highly developed ciliary structure, and its motility may be necessary for removing invasive microorganisms, guiding fertilization, and promoting zygote movement, which may be affected by pathogens (Barton et al., 2020). For instance, the fine cilia on ciliated cells are vulnerable to invasion and destruction by pathogens such as Neisseria gonorrhoeae, Mycoplasma, Chlamydia, and others, resulting in edema, necrosis, functional decline or loss of cilia (Barton et al., 2020). Yang et al. established a mice model of Chlamydia infection and observed the activation of complement factor 5 (C5) in the hydrosalpinx group, characterized by fallopian tube enlargement and fluid accumulation (Yang et al., 2014). Previous studies have suggested that C3 and C5 may initiate fibrotic responses in epithelial cells (Portilla and Xavier, 2021). Ciliary lesions and fibrosis cause inflammatory thickening of the fallopian tube wall and narrowing or even blockage of the lumen, which may lead to adverse reproductive outcomes such as tubal infertility and tubal pregnancy (Barton et al., 2020; Yang et al., 2014; Portilla and Xavier, 2021). Furthermore, animal experiments showed that Lactobacillus rhamnosus GG attenuated the pathological damage caused by Chlamydial muridarium infection in the fallopian tube (Zhou et al., 2021). This study suggests that Lactobacillus may help maintain the homeostasis of female fallopian tube tissue. However, the underlying interaction mechanisms between Lactobacillus and the histological and immune systems in the fallopian tube have been poorly studied.

The vaginal microbiome varies among women of different races. In terms of the vaginal microbial species, anaerobic bacteria tend to colonize the reproductive tract of White women, Asian women and Caucasian women, while Candida is more prevalent in colonizing the vaginal mucosa of Black women (Gupta et al., 2020). Similarly, the infection rates of Mycoplasma, Ureaplasma urealyticum and Neisseria gonorrhoeae were higher in Black women, and the separation rate of Trachomatis in the vagina in Black women and Hispanic women was significantly higher than that in White women (Newton et al., 2001). In terms of the vaginal microbial diversity, Black women and Hispanic women had significantly higher diversity than Asian women and White women (Ravel et al., 2011). In terms of the abundance of vaginal microbes, the vaginal microbiome dominated by Lactobacillus was found in White women (89.7%), Asian women (80.2%), Black women (61.9%) and Hispanic women (59.6%), and the abundance of Lactobacillus was only 37% in Black women in another study (Ravel et al., 2011; Anahtar et al., 2015). Other studies have found that BVAB in the vagina of Black women is 25.8% higher than that of White women, which may be linked to the increase in corticotropin-releasing hormone-related gene mutations (Newton et al., 2001; Ryckman et al., 2009). Currently, the specific reasons for the race-related differences in the vaginal microbiome are unknown. Generally, the differences may be related to host genetic characteristics, geographic environments, and lifestyles. Therefore, studying the effects of race on the microbiome requires further consideration of the mixed effects of additional variables, which can help to develop personalized microecological therapies.

The reproductive tract microbiome changes with the age of women and interacts with women throughout the lifespan. In infancy, the vaginal microbiome is dominated by a mixture of aerobic bacteria and anaerobic bacteria. Before puberty, anaerobic microbial communities dominate the vaginal microbiome. During puberty and childbearing age, the vaginal microbiome changes to be dominated by Lactobacillus (Ravel et al., 2011; Song et al., 2020). After menopause, the abundance of Lactobacillus in the vaginal microbiome gradually decreases, while that of Escherichia coli increases, leading to menopausal symptoms such as vaginitis and vaginal dryness (Marnach et al., 2022). Another study showed that uterine microbial diversity decreased with increasing age, while vaginal microbial diversity increased (Wang et al., 2021). The compositions of the vaginal microbiome and uterine microbiome underwent some changes with age, which may be related to estrogen levels. Overall, these findings suggest that age is a significant factor affecting the female reproductive tract microbiome and has an important impact on reproductive health. Exploring the relationship between age and the female reproductive tract microbiome could help alleviate age-related reproductive diseases.

Pregnancy is an important window for regulating the vaginal microbiome, and the effect of it has been proven. ① The diversity and richness of the vaginal microbiome in pregnant women are more stable in pregnant women than in nonpregnant women (Romero et al., 2014). ② In the early stage of pregnancy, Lactobacillus dominates the vaginal microbiome and persists throughout pregnancy. The abundance of Lactobacillus increases with gestational age, which may be related to changes in SCFA and hormone levels (Jost et al., 2014). ③ Compared with preterm women, term women have a richer vaginal microbial diversity with a higher abundance of Lactobacillus (except for L. iners) and a lower abundance of anaerobic bacteria (Aagaard et al., 2012; Lewis et al., 2017). ④ The vaginal microbiome undergoes significant alterations after normal delivery due to decreased estrogen levels and lochia discharge; this leads to a decrease in communities characterized by Lactobacillus and an increase in the diversity of anaerobic bacteria such as Anaerococcus, Prevotella, and Peptoniphilus, which may last up to one year postdelivery (DiGiulio et al., 2015; Severgnini et al., 2022). It is worth noting that changes in the vaginal microbiome during late pregnancy aid in increasing maternal energy storage, thus contributing to fetal growth and development (Wang et al., 2020). Accurately understanding the changes in the maternal vaginal microbiome during pregnancy can better ensure favorable maternal pregnancy outcomes and fetal health.

The mode of delivery (cesarean section or vaginal delivery) affects the gut microbiome of infants, and emerging research has also found its impact on the vaginal microbiome in adulthood (de Koff et al., 2022; Stennett et al., 2020). The relative abundance of L. jensenii and L. iners was higher in women who delivered vaginally, while the abundance of Prevotella bivia was higher in women who delivered by cesarean section (Stennett et al., 2020). Moreover, compared with women who delivered vaginally, women who delivered by cesarean section exhibited a threefold increase in the likelihood of having low-Lactobacillus CST IV. The elevation in this risk heightens susceptibility to sexually transmitted infections (STIs) and abnormal pregnancy outcomes (Stennett et al., 2020). The composition and relative abundance of Lactobacillus and other microbes in the vagina varied between women who underwent cesarean delivery and those who delivered vaginally, suggesting that vaginal delivery is an important method to enhance the reproductive tract health of the baby into adulthood.

The composition of the vaginal microbiome and endometrial microbiome are closely correlated with parity. Kervinen et al. reported that parity was inversely associated with the abundance of L. crispatus. The relative abundance of L. crispatus exhibited a declining trend, with percentages of 58.1% in nulliparous women, 25.7% in women who had given birth once, and 15.4% in women who had given birth twice or more (Kervinen et al., 2022). Additionally, the relative abundances of L. gasseri and L. iners increased with higher parity (Kervinen et al., 2022). This study suggested that nulliparity was a significant contributing factor to the high abundance of L. crispatus associated with gynecological health. Similarly, Bogado et al. found that the abundences of Fusobacterium and Bacillus in the endometrial microbiome of cows were lower in multiparous cows than in nulliparous cows, while the abundences of Bifidobacterium and Staphylococcus were higher in multiparous cows (Bogado Pascottini et al., 2021). As a result, parity should be taken into account when studying the composition of the vaginal and endometrial microbiome and its effect on reproductive outcomes.

The vaginal pH affects the composition of the vaginal microbiome, and in the population with a mean vaginal pH greater than 4.7, Lactobacillus is no longer the dominant microbiome (Ravel et al., 2011; Barrientos-Durán et al., 2020). Moreover, excessive changes in pH cause disorders of the vaginal microbiome, placing the body in a pathological state. For instance, vaginal irrigation can frequently disrupt the homeostasis of the microbiome, increase the vaginal pH, inhibit the growth of Lactobacillus, and lead to external bacterial invasion (Gajer et al., 2012). Vaginal irrigation can not only introduce exogenous substances that cause pH changes but can also promote mechanical cleaning of symbiotic bacteria, thus affecting the ecological balance of the vagina (Brotman et al., 2008). Notably, when studying the composition of the vaginal microbiome, it is necessary to consider the adaptive changes in the composition and types of female reproductive tract microbiome under different vaginal pH values.

As hormone levels change during the menstrual cycle, the microbiome composition from the vagina to the uterus changes; compared to the secretory phase, the proliferative phase seems to be associated with bacterial proliferation in the vagina and endometrium, and the microbiome composition is less stable during this phase (Romero et al., 2014; Sola-Leyva et al., 2021). Shen et al. found that estrogen therapy in postmenopausal women with atrophic vaginitis significantly increased the relative abundance of Lactobacillus in the vagina (Shen et al., 2016). Additionally, the use of hormonal contraception (e.g., long-acting hormonal contraceptives, intrauterine devices) may have an adverse impact on the female reproductive tract microbiome, resulting in invasion and colonization by anaerobic microbes and Actinomycetes, and their long-term use can put women at greater risk of bacterial vaginosis (BV) and streptococcal infection (Vodstrcil et al., 2013; Whitney et al., 2021; Aubert et al., 1980). Taken together, the effects of hormones on the female reproductive tract microbiome should not be ignored and need to be further studied. Resolving this issue will contribute to regulating the female reproductive tract microbiome using hormones, reducing the occurrence of hormone-mediated female reproductive tract diseases, and promoting female reproductive health.

Dietary habits are a complex psychosocial behavior that can easily be confused by socioeconomic factors. Despite this, it has long been considered that diet is related to the composition and function of the gut microbiota (Tilg, 2010). The gut microbiota glycolyses and ferments indigestible carbohydrates in the diet into short-chain fatty acids (SCFAs) (Amabebe and Anumba, 2020). Considering that the gut microbiota continues to migrate to the vagina and SCFAs may be transmitted to the vagina through the bloodstream, it is evident that diet has an impact on the vaginal microbiota (Amabebe and Anumba, 2020). Subsequent studies have shown that inadequate intake of dietary micronutrients, such as vitamins A, C, and E and β-carotene, as well as a high glycemic load in women of childbearing age are associated with an increased incidence of BV (Tohill et al., 2007). Conversely, the intake of betaine and folic acid can mitigate the risk of BV, and a high-starch diet may promote vaginal health by lowering the vaginal pH (Miller et al., 2016; Tuddenham et al., 2019). In summary, a good diet is vitally important for a healthy female reproductive tract microbiota, which contributes to maintaining reproductive health and improving reproductive capacity.

Antibiotics are inevitable in the fight against reproduction tract infections, and their use can suppresses the propagation of pathogenic bacteria and the development of diseases. In addition, the use of antibiotics during pregnancy can cause changes in the maternal reproductive tract microbiome, which have long-term effects on early neonatal microbial colonization (Romero et al., 2014). Overuse of antibiotics disrupts the normal reproductive tract microbiome, leading to the emergence of a large number of drug-resistant microorganisms (DRMs). Marnach et al. found that the overuse of antibiotics easily caused vaginal Candida infection, and the longer the use time was, the higher the probability of infection (Marnach et al., 2022). Overall, the rational use of antibiotics is a factor that must be considered to maintain reproductive health. Further research into how antibiotics interact with the reproductive tract microbiome will provide valuable insights into the use of antibiotics, which may help mitigate the negative effects of antibiotics.

The transmission of beneficial microbiome occurs during sexual activity, which may confer advantages to both males and females (Noyes et al., 2018; Smith and Mueller, 2015). Not only is the dominance of Gardnerella vaginalis in women is significantly associated with reproductive tract inflammation in male partners, but other studies have confirmed that inappropriate sexual behavior can also seriously interfere with the balance of reproductive tract microbiome in women of childbearing age (Vodstrcil et al., 2017; Mändar et al., 2015). Kreisel et al. proposed that the incidence of sexually transmitted diseases is highest among women and man aged 15 to 24 in the United States, possibly because women and man in this age group may have unclean, active and excessive sexual lives (Kreisel et al., 2021). BV can be sexually transmitted from a women to a man and his female partner, and the incidence in women with more than 2 sexual partners was 1.77 times higher than that in women with only 1 sexual partner or no sexual partner (Chen et al., 2017; Faught and Reyes, 2019). Engaging in sexual activity with multiple sexual partners can destabilize the vaginal microbiome and reduce the relative abundance of Lactobacillus, thereby increasing the risk of BV transmission (Gajer et al., 2012; Jespers et al., 2015). Semen is an alkaline substance, and sexual intercourse will cause a temporary increase in the vaginal pH, which can be restored 8 hours after intercourse. If sexual intercourse occurs repeatedly within a day, the vaginal pH is always high, which is conducive to the growth of conditional pathogens (Tuddenham et al., 2021). Therefore, proper sexual habits are an important factor in maintaining reproductive tract health.

The composition of the vaginal microbiome may be influenced by the type of sanitary napkins used, and improper usage habits of sanitary napkins can lead to the colonization of pathogens in the reproductive tract. Hickey et al. compared the changes in the vaginal microbiome in mid-cycle menstrual and menstrual women by using two designated types of sanitary napkins. They found that neither type of sanitary napkins had a significant effect on the composition of the vaginal microbiome at different stages of the menstrual cycle (Hickey et al., 2013). Other studies have demonstrated that there were statistically significant differences in the prevalence of Gardnerella vaginalis and anaerobic gram-negative rods between the groups using the two different types of sanitary napkins during the premenstrual visit (Chase et al., 2007). Hence, it is necessary to control for the confounding factor of sanitary napkin brands when investigating the changes in the vaginal microbiome during menstruation. The use of sanitary napkin products during nonmenstrual periods may impede perineal air circulation, elevate local temperature and humidity, and potentially alter vaginal pH levels, thereby promoting the colonization and proliferation of Candida and Staphylococcus aureus (Marnach et al., 2022; Moosa et al., 2020). During the menstrual period, menstrual blood flows out of the vagina, and the congealed blood is a good culture medium for bacteria. At this time, if sanitary napkins with unqualified disinfection ratings are used or are used for too long, exogenous pathogenic bacteria are likely to reproduce in the menstrual blood and invade the reproductive tract (Zhang et al., 2021).

Exercise and sufficient sleep promote microbial homeostasis by reducing inflammatory responses, while poor modern lifestyles disrupt the balance of the reproductive tract microbiome. Drying clothes in poorly ventilated environments or wearing synthetic underwear, can lead to an imbalance in the microbial community, particularly Candida proliferation (Marnach et al., 2022). The rate of vaginal Candida colonization in women using vaginal irrigation fluid (26%) was higher than that in women without this habit (20%), and excessive vaginal irrigation may lead to vaginal microbiome imbalance, increasing the incidence of upper reproductive tract infections (Hyman et al., 2014; Barousse et al., 2004). Excessive cleaning of the vulvar area with potential irritants such as soaps, bubble baths, powders, or vaginal sprays can also affect the vaginal microbiome (Watson and Calabretto, 2007). In addition, smokers have a lower proportion of vaginal Lactobacillus, and smoking can increase the incidence of BV (Ding and Schloss, 2014; Brotman et al., 2014; Fettweis et al., 2014). Strengthening reproductive health education and developing a set of healthy lifestyle schemes for the entire population, are beneficial to maintaining the homeostasis of the reproductive tract microenvironment and improving health indicators.