Mechanisms and Therapeutic Implications of Microbiome‐Mediated Immune Dysregulation in Recurrent Pregnancy Loss and Implantation Failure

ABSTRACT

Problem

Recurrent pregnancy loss (RPL), defined as two or more consecutive pregnancy losses before 20 weeks of gestation, affects 1%–5% of couples of reproductive age worldwide. Growing evidence indicates a role for the microbiome in reproductive health, particularly in unexplained RPL.

Method of Study

Based on a review of literature from PubMed, EMBASE, and Web of Science databases from January 2020 to September 2025, this comprehensive overview explores the current understanding of the link between microbiome dysbiosis and RPL.

Results

Microbiome dysbiosis, especially a reduction in Lactobacillus dominance and increased diversity, is strongly linked to RPL across multiple reproductive sites. RPL is associated with the loss of protective Lactobacillus crispatus and a higher presence of potentially harmful bacteria, including Gardnerella vaginalis and Atopobium vaginae. An altered gut microbiome, particularly with lipopolysaccharide‐producing gram‐negative bacteria, contributes to systemic inflammation and immune dysfunction by disrupting maternal‐fetal immune tolerance. The microbiome‐immune axis is essential for establishing maternal‐fetal tolerance, with dysbiosis promoting pro‐inflammatory Th1/Th17 responses while suppressing regulatory T cells. Multiple mechanisms connect microbiome dysbiosis to RPL, including local inflammation, systemic immune issues, disruption of maternal‐fetal immune tolerance, molecular mimicry, and autoimmunity.

Conclusions

The microbiome is a promising new target for RPL treatment, with personalized microbial profiling and targeted therapies showing potential to improve pregnancy outcomes. Clinical implementation requires standardized protocols, larger randomized controlled trials, and validation of microbiome‐targeted interventions.

1. Introduction

Recurrent pregnancy loss (RPL), defined as two or more consecutive pregnancy losses before 20 weeks of gestation, affects 2%–5% of couples attempting conception, with rates varying by population and diagnostic criteria [1]. Major causes include chromosomal anomalies (50%–60% of sporadic losses), uterine structural abnormalities in 10%–20% of women, endocrine disorders like thyroid dysfunction and PCOS, and autoimmune conditions such as antiphospholipid syndrome [2, 3]. Modifiable risk factors include advanced maternal age (> 35 years), obesity, smoking, and alcohol intake [4, 5]. Despite standardized evaluation protocols, over half of RPL cases remain unexplained [6]. Emerging research indicates that changes in vaginal and Endometrial microbiome composition contribute to RPL development through disrupted inflammatory responses, immune system issues, and impaired implantation [7, 8].

Adverse pregnancy complications, including RPL, are influenced by genetic predisposition, environmental factors, and the human microbiome [5]. This complex microbial ecosystem consists of diverse microorganisms inhabiting specific anatomical sites with distinct compositions and plays essential roles in nutrient metabolism, immune regulation, inflammation control, and mucosal defense [5, 9]. The local microbiome of the female reproductive tract, particularly the vaginal and endometrial microbiomes, can significantly impact fertility, embryo implantation, and pregnancy maintenance (Figure 1). Disrupted microbial homeostasis, known as dysbiosis, has been linked to pregnancy complications including preterm birth, miscarriage, and RPL [10]. This review explores the role of vaginal, endometrial, and gut microbiomes in RPL, where alterations in microbiome composition marked by an overgrowth of potentially pathogenic bacteria are associated with adverse pregnancy outcomes.

FIGURE 1.

The role of microbiome dysbiosis in reproductive failure. The gut, vaginal, and endometrial microbiomes interact to maintain reproductive tract homeostasis. Dysbiosis within the vaginal and/or endometrial microbiomes disrupts immune tolerance at the maternal‐fetal interface, thereby increasing the risk of recurrent pregnancy loss and implantation failure.

2. Methods

A thorough literature search was conducted using PubMed, EMBASE, and Web of Science databases from January 2020 to September 2025. Search terms included combinations of “microbiome,” “dysbiosis,” “recurrent pregnancy loss,” “miscarriage,” “Lactobacillus,” and “endometrial microbiota.” Studies were included if they examined microbiome composition in relation to RPL in human subjects and were published in English. Case reports, conference abstracts, and studies with fewer than 10 participants were excluded. Data extraction focused on study design, population characteristics, microbiome analysis methods, and clinical outcomes. Quality assessment centered on the appropriateness of study design, sample size sufficiency, microbiome analysis methodology, and statistical robustness. Due to the heterogeneity of study designs and outcome measures, a qualitative synthesis approach was adopted rather than a meta‐analysis.

3. An Overview of the Human Microbiome

The human microbiome is established early in life, influenced by factors such as delivery mode, feeding practices, antibiotic use, and environmental conditions, and has become a key factor in both health and disease [10, 11]. The dominance of beneficial bacteria, especially Lactobacillus, in the female reproductive tract (eubiosis), is linked to better implantation and ongoing pregnancy [11].

3.1. Gut Microbiome

The gut microbiota is the largest microbial reservoir in the human body, containing up to 10^{1 4} microorganisms in adults, collectively harboring approximately 100–150 times more genes than the human genome [12]. It exerts systemic effects on immune function, metabolism, and inflammation, including immune system development, pathogen resistance, maintenance of epithelial barrier integrity, and nutrient metabolism [9, 13]. Its functions include fermenting indigestible polysaccharides, producing short‐chain fatty acids (SCFA), synthesizing vitamins, and metabolizing xenobiotics [14, 15]. These functions depend on site‐specific microbial compositions that vary significantly between different anatomical locations [14, 16].

During pregnancy, the gut microbiome undergoes significant compositional changes, with an increased abundance of Firmicutes and Proteobacteria, and a decrease in microbial diversity [17]. Reduced gut microbial diversity, coupled with the relative abundances of Prevotella, Prevotellaceae UCG‐003, and Selenomonas, were reported in miscarriage cases [18]. The gut microbiome alterations in RPL are characterized by increased abundance of gram‐negative bacteria capable of producing lipopolysaccharides (LPS), which in turn trigger systemic inflammatory responses, leading to pregnancy failures [19, 20].

3.2. Endometrial Microbiome

Although less diverse than the gut microbiome, the urogenital microbiome plays an essential role in reproductive physiology. While the endometrial microbiome contains significantly fewer bacteria (10²–10⁴) than the vaginal microbiome (10⁸–10⁹), it is a functionally balanced microbial ecosystem dominated by Lactobacillus species, associated with improved embryo implantation, immune tolerance, and positive pregnancy outcomes [21, 22]. Studies have documented unique endometrial microbiome patterns in women with RPL, characterized by comparatively higher levels of Acinetobacter, Anaerobacillus, Erysipelothrix, Bacillus, and Hydrogenophilus species compared to controls [11, 20]. The dominance of Lactobacillus iners over Lactobacillus crispatus in endometrial samples from women with RPL has been linked to endometrial dysbiosis and negative reproductive outcomes [11, 23]. This shift is biologically significant, as Lactobacillus iners cannot produce D‐lactic acid and bacteriocins that are characteristic of more protective Lactobacillus species [23].

3.3. Vaginal Microbiome

Once considered sterile, the female reproductive tract hosts diverse microbial communities essential to reproductive health and pregnancy establishment [24]. The vaginal microbiome is generally dominated by Lactobacillus species, mainly Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, and Lactobacillus iners, which interact with vaginal epithelial cells and immune components. They promote mucosal barrier integrity by maintaining acidic pH (3.8–5.0) and producing antimicrobial compounds such as lactic acid, hydrogen peroxide, and bacteriocins [25, 26]. The cervical microbiome functions as a transitional zone with moderate diversity between the vaginal and endometrial environments [7], while the endometrial microbiome influences implantation and pregnancy support, with Lactobacillus dominance associated with positive outcomes [8]. The urinary microbiome includes diverse bacterial communities that contribute to urinary tract health [27]. Microbial imbalance across these sites may lead to adverse pregnancy outcomes, such as RPL [11, 18].

Vaginal microbiomes are generally classified into five community state types (CSTs) [28]. CST I‐III are dominated by different Lactobacillus species: Lactobacillus crispatus (CST I), Lactobacillus gasseri (CST II), and Lactobacillus iners (CST III) [25, 29]. CST IV is characterized by diverse anaerobic bacterial communities with low Lactobacillus levels, including Gardnerella, Atopobium, Prevotella, and Mobiluncus, which are divided into CST IV‐A and CST IV‐B [30]. CST IV is often linked to bacterial vaginosis, increased mucosal inflammation, and negative reproductive outcomes, with a shift toward the pro‐inflammatory CST IV profile associated with chronic endometritis, a known risk factor for implantation failure and miscarriage [31]. CST V is dominated by Lactobacillus jensenii and is linked to a lower risk of bacterial vaginosis, commonly found in Hispanic and Black women, and may change during hormonal fluctuations [28].

4. Microbiome Dysbiosis and Adverse Pregnancy Complications

The composition of the endometrial microbiome is crucial for successful implantation and favorable pregnancy outcome [8, 32]. Lactobacillus‐dominated endometrial communities are associated with higher implantation and live birth rates in assisted reproductive technology cycles [32, 33], while non‐Lactobacillus‐dominated microbiomes correlate with poorer reproductive outcomes [21, 34]. These findings suggest microbial dysbiosis contributes to pregnancy complications, including recurrent loss [34, 35].

4.1. Miscarriage and Early Pregnancy Loss

Emerging evidence has documented a complex relationship between disruptions in the normal composition and function of the body microbiome, known as dysbiosis, across multiple anatomical sites and early pregnancy loss [35, 36]. Altered vaginal, endometrial, gut, and oral microbiomes have been linked to pregnancy loss through inflammatory cascades, immune dysfunction, and metabolic disruption [37]. These associations vary across populations [38], offering targeted therapeutic opportunities.

4.2. Recurrent Pregnancy Loss (RPL)

Women experiencing recurrent implantation failure and RPL often show significant changes in vaginal microbiome composition, marked by a loss of Lactobacillus dominance and an increase in potentially harmful bacteria, including Gardnerella vaginalis [11, 21, 39]. Women with RPL tend to have fewer beneficial Lactobacillus species, especially Lactobacillus crispatus, and higher levels of Gardnerella vaginalis, Atopobium vaginae, and anaerobic bacteria [39]. This vaginal imbalance is linked to increased local inflammation and elevated pro‐inflammatory cytokines, which can lead to pregnancy loss [40].

An earlier study involving 35 women with recurrent implantation failure documented up to 90% reduction in Lactobacillus dominance, accompanied by marked increases in Streptococcus, Staphylococcus, and Gardnerella [8]. A recent multi‐center study of 342 infertile women from diverse ethnicities reported dysbiotic endometrial microbiota comprising Atopobium, Bifidobacterium, Chryseobacterium, Gardnerella, Haemophilus, Klebsiella, Neisseria, Staphylococcus, and Streptococcus associated with unsuccessful reproductive outcomes [8]. Subsequent Finnish and Chinese studies confirmed links between dysbiotic endometrial microbiota and poor reproductive outcomes, evidenced by significant Lactobacillus reduction and increased pathogenic bacteria [41, 42]. Antibiotic treatment improved clinical pregnancy (50.4% vs. 37.5%) and ongoing pregnancy (42.4% vs. 25%) in the dysbiotic group [42]. Japanese women with unexplained RPL showed cervical Cutibacterium and Anaerobacillus strongly linked with subsequent miscarriage, with an adjusted odds ratio of 16.90 [43].

Dysbiosis in the gut has been linked to autoimmune diseases, including type 1 diabetes and multiple sclerosis, primarily through modulation of T regulatory cells, systemic inflammation, and alterations in microbial metabolites such as SCFAs [44]. Emerging evidence suggests a similar impact on pregnancy outcomes. Gut microbiome dysbiosis in women experiencing unexplained pregnancy loss reportedly impairs pregnancy maintenance by triggering systemic inflammatory cascades driven by cytokine network imbalances [18]. Recent reviews have highlighted the role of dysbiosis in RPL through heightened inflammatory responses and disrupted insulin signaling [45]. Women with RPL exhibit reduced levels of SCFA‐producing bacteria, including Faecalibacterium prausnitzii and Roseburia spp., as well as increased proportions of pro‐inflammatory taxa such as Proteobacteria, Fusobacterium, and Clostridium perfringens [18, 37, 46]. Furthermore, the estrobolome, representing gut microbial genes involved in estrogen metabolism, may influence estrogen levels and subsequently affect vaginal microbiome composition and endometrial proliferation [47].

4.3. Preterm Birth and Other Complications

Emerging evidence links microbiome dysbiosis at various body sites to an increased risk of pregnancy complications, including preterm birth, intrauterine growth restriction, and preeclampsia [48, 49]. A large study of 12,000 samples from mainly African women showed that preterm birth was associated with reduced Lactobacillus crispatus and increased BVAB1, Sneathia amnii, TM7‐H1, and Prevotella spp. [48]. Meta‐analyses confirmed that community state types with low Lactobacillus were linked to a higher risk of preterm birth, while Lactobacillus crispatus remained protective across populations [50]. Racial disparities were evident, with African American women exhibiting greater microbiome instability [51]. Variability in the vaginal microbiome early in pregnancy is associated with first‐trimester risk, whereas taxa such as Olsenella and Clostridium sensu stricto tend to increase later [51, 52]. Proposed mechanisms include ascending infection, cytokine upregulation, disruption of the cervical barrier, and activation of premature labor [53].

4.4. Uterine Microbiome and Endometrial Receptivity

Once considered sterile, the uterine cavity is now recognized to host low‐biomass microbial communities detectable by next‐generation sequencing [7]. Although the uterine microbiome remains less well‐characterized than the vaginal microbiome, growing evidence suggests that a Lactobacillus‐dominated endometrial environment supports embryo implantation and endometrial receptivity [21]. In contrast, increased abundance of non‐Lactobacillus taxa, particularly Proteobacteria, Firmicutes, and Bacteroidetes, has been associated with impaired receptivity and heightened inflammatory responses [21]. Chronic endometritis, frequently subclinical, is defined by the histological presence of plasma cells in the endometrial stroma and is more prevalent in women with RPL [54], strongly linked to endometrial dysbiosis characterized by overgrowth of Escherichia coli, Enterococcus faecalis, Streptococcus agalactiae, and other pathogens [54]. Antibiotic treatment targeting these infections has improved reproductive outcomes in affected women [31].

5. Mechanisms Linking Microbiome Dysbiosis to RPL

Growing evidence indicates that microbiome dysbiosis plays a critical role in RPL through several distinct yet interconnected mechanisms. These mechanisms affect endometrial receptivity and implantation, compromise the maternal‐fetal barrier, and alter systemic immune responses. Elucidating these pathways is essential for identifying novel biomarkers and therapeutic targets aimed at improving pregnancy outcomes in women affected by RPL. Specifically, four primary mechanisms have been identified in the dysbiosis‐RPL relationship, often acting synergistically to undermine reproductive success. A deeper understanding of these interactions will be key to advancing targeted interventions and personalized treatment strategies.

5.1. Local Inflammatory Response

Vaginal and endometrial microbial dysbiosis in the reproductive tract can trigger local inflammatory responses that impair endometrial receptivity and embryo implantation [11, 33]. This involves activation of innate immune responses, particularly Toll‐like receptors (TLRs), which identify pathogen‐associated molecular patterns, such as lipopolysaccharide from Gram‐negative bacteria [55]. TLR activation leads to increased production of pro‐inflammatory cytokines, including TNF‐α, IL‐1β, and IL‐6, weakening the mucosal environment essential for proper implantation [11, 56]. Sneathia and Gardnerella species, often enriched in dysbiotic microbiomes, cause epithelial damage and inflammation that hinder implantation [21]. These pro‐inflammatory changes reduce the expression of implantation‐related markers, such as leukemia inhibitory factor and integrins, further impairing trophoblast adhesion and invasion [57]. This highlights a direct role of local microbial dysbiosis in disrupting conditions necessary for successful embryo implantation [40].

5.2. Systemic Immune Dysfunction

The gut microbiome exerts profound effects on systemic immune function through the production of microbial components and metabolites, such as SCFAs, LPS, and tryptophan derivatives, that enter systemic circulation and modulate immune responses [44]. Correlation analyses indicate that certain microbe‐associated metabolites are positively associated with elevated Th1/Th17 cytokine levels in women experiencing miscarriage, suggesting a pro‐inflammatory systemic immune shift [18]. One key mechanism involves the translocation and systemic dissemination of LPS, driven by increased intestinal permeability. Also known as “leaky gut”, this was associated with disrupted tight junction proteins, including claudins, which facilitate the translocation of bacterial components into circulation [29], leading to activation of TLR4 signaling, promoting inflammation and impairing maintenance of pregnancy [11]. In addition, altered SCFA levels were linked to regulatory T‐cell (Treg) dysfunction, further disrupting maternal immune tolerance in early pregnancy (Figure 2).

FIGURE 2.

Dysbiosis‐driven impairment of implantation. In healthy reproductive tract (left), balanced microbiome supports endometrial receptivity through expression of implantation markers including leukemia inhibitory factor (LIF) and integrins. In dysbiotic conditions (right), pathogenic bacteria such as Sneathia and Gardnerella activate toll‐like receptors (TLRs) through lipopolysaccharide (LPS) recognition, triggering inflammatory cascade involving TNF‐α, IL‐1β, and IL‐6, ultimately reducing implantation marker expression and endometrial receptivity.

5.3. Disruption of Maternal‐Fetal Immune Tolerance

Successful pregnancy requires the establishment of maternal‐fetal immune tolerance, characterized by a shift from (pro‐inflammatory) Th1 responses toward (anti‐inflammatory) Th2 responses and the expansion of Treg populations, which suppress fetal‐directed immune responses [3, 58]. Also included are the tolerogenic uterine NK (uNK) cells, which are accountable for implantation and placental development [59]. Microbiome dysbiosis, particularly in the gut and reproductive tract, disrupts this delicate immune balance by promoting pro‐inflammatory responses while suppressing regulatory mechanisms. Studies show that IL‐2, IL‐17A, IL‐17F, TNF‐α, and IFN‐γ are significantly increased in the serum of women with miscarriage, indicating a systemic inflammatory environment that undermines immune tolerance [18, 52].

Recent evidence indicates that the vaginal and endometrial microbiota critically influence the induction of paternal antigen‐specific regulatory T cells (Tregs), which are essential for maternal‐fetal tolerance. A healthy, Lactobacillus‐dominated microbiota fosters a local anti‐inflammatory milieu by enhancing epithelial expression of key tolerogenic mediators, TGF‐β and indoleamine 2,3‐dioxygenase (IDO), which drive peripheral Treg differentiation [11, 24, 40]. In contrast, dysbiotic communities enriched in Gardnerella, Atopobium, Sneathia, and related species augment pro‐inflammatory cytokines, including IL‐6 and IL‐17 [11, 21, 40], thereby suppressing Treg stability and favoring Th17 cell polarization [11, 21, 40]. Endometrial dysbiosis can also disrupt dendritic cell‐T cell crosstalk, limiting tolerogenic antigen presentation and weakening systemic immune adaptation to pregnancy [11, 22, 24, 40]. This provides a biologically plausible link between reproductive tract microbiome dysregulation and the failure of immunological tolerance during early gestation.

Uterine natural killer (uNK) cells and Tregs, along with T‐helper subsets, are critical for immune tolerance at the maternal‐fetal interface. Accounting for 70%–80% of decidual lymphocytes in early gestation, uNK cells facilitate placental development by secreting angiogenic and immunomodulatory cytokines, thereby promoting trophoblast invasion and spiral artery remodeling [59]. Dysbiosis in the vaginal or endometrial microbiota reportedly disrupts uNK cell activation via pattern recognition receptor signaling, leading to excessive cytotoxicity and impaired placentation [11, 40, 55]. In parallel, Tregs maintain tolerance to paternal alloantigens by suppressing pro‐inflammatory Th1 and Th17 responses [58]. These consistent reductions in Treg number and/or function were demonstrated in women with recurrent miscarriage [58, 59]. In addition, microbiome‐derived SCFAs promote extrathymic Treg differentiation [60]; consequently, reduced SCFA‐producing bacteria may compromise peripheral Treg induction [18, 44]. This demonstrates that perturbations in the microbiome‐uNK‐Treg axis shift the maternal immune balance toward inflammation, thus increasing the risk of pregnancy loss.

5.4. Molecular Mimicry and Autoimmunity

Emerging evidence suggests that microbiome dysbiosis may trigger autoimmune responses implicated in RPL through molecular mimicry [44, 61], particularly in individuals who are genetically predisposed [44]. Certain microbial antigens share structural or sequential homology with host proteins, leading the immune system to target self‐antigens mistakenly [44]. Escherichia coli, Mycoplasma hominis, and other bacterial species have been shown to express proteins with epitopes that mimic human phospholipid‐binding proteins, triggering the production of antiphospholipid antibodies (aPLs) [44, 62]. Commonly observed in antiphospholipid syndrome (APS), these aPLs are linked with impaired placental development, thrombotic events, and pregnancy loss [2, 63]. Furthermore, microbial structural components and metabolites, including peptidoglycan, reportedly modulate dendritic cell function and promote the activation of self‐reactive T and B cells [64]. Collectively, these findings support a role for microbiome‐mediated molecular mimicry in the development of autoimmunity contributing to RPL (Figure 3).

FIGURE 3.

Linking microbiome dysbiosis to RPL via molecular mimicry. Dysbiosis‐associated microbes (E. coli) present antigens that resemble host proteins, leading to molecular mimicry and the activation of autoreactive T and B cells. This results in the generation of antiphospholipid antibodies, which consequently contribute to impaired placentation, inflammatory injury at the maternal‐fetal interface, and ultimately RPL.

6. Microbiome‐Based Diagnostics

Endometrial Microbiome Metagenomic Analysis (EMMA) and Analysis of Infectious Chronic Endometritis (ALICE) are diagnostic tools developed to assess reproductive tract dysbiosis and its impact on fertility [8, 65]. EMMA classifies patients based on the endometrial microbiome composition, with a Lactobacillus relative abundance exceeding 90% considered the threshold for a Lactobacillus‐dominated profile [65]. LD microbiomes are consistently associated with better reproductive outcomes, including higher implantation and pregnancy rates [7, 8, 21]. ALICE detects chronic endometritis by identifying specific pathogens such as Escherichia coli, Enterococcus faecalis, and Streptococcus species through targeted metagenomic sequencing [8, 65].

Recognized by the presence of plasma cells in the endometrial stroma, chronic endometritis (CE) is increasingly acknowledged as a contributing factor to RPL, with a prevalence of 29.6% in women with RPL compared to 6.8% in fertile controls [66, 67]. In this regard, higher relative dominance of Lactobacillus iners and positivity of Ureaplasma species were reported in women with CE [66]. Furthermore, microbial profiling is a promising diagnostic tool for RPL, with advanced sequencing technologies enabling comprehensive assessment of microbial communities across multiple sites for personalized risk stratification and treatment planning [66, 68].

EMMA and ALICE are PCR‐based assays that quantify key endometrial taxa with high sensitivity, speed, and low cost. However, they are limited to predefined panels and miss rare or uncharacterized microbes. In contrast, next‐generation sequencing (NGS), including 16S rRNA and shotgun metagenomics, offers broader, culture‐independent profiling with functional insights. However, it is more resource‐intensive and vulnerable to contamination in low‐biomass samples. PCR and NGS serve as complementary tools, balancing targeted clinical specificity with ecological breadth [6, 11, 22]. Beyond EMMA and ALICE, the uterine microbiota is assessed using targeted PCR or NGS [65]. PCR enables rapid, cost‐effective detection of predefined pathogens but has a limited taxonomic scope. NGS provides comprehensive, culture‐independent profiling of dominant and rare taxa with functional insights [69], though it requires strict contamination control and advanced bioinformatics [65, 69]. Comparative studies show PCR is suitable for pathogen screening or chronic endometritis diagnosis, while NGS reveals broader microbial diversity and reproductive relevance [11, 22, 64].

7. Microbiome‐Targeted Therapeutic Interventions

Targeted antibiotic therapy is the first‐line treatment for women with documented pathogenic bacteria or chronic endometritis. Administering treatment before embryo transfer improves endometrial microbial health and increases live birth rates [31, 54]. Standard regimens include doxycycline for Chlamydia and Ureaplasma, metronidazole for anaerobes, and ciprofloxacin for gram‐negative pathogens. However, antibiotic therapy should be followed by probiotic supplementation to restore beneficial communities [54, 70]. Probiotic supplementation shows promise, with daily L. salivarius CECT5713 for six months achieving 56% successful pregnancy rates [68]. Beneficial strains include Lactobacillus crispatus for restoring the vaginal acidic environment, Lactobacillus rhamnosus for immunomodulatory effects, and multi‐strain formulations for comprehensive restoration [70]. Vaginal microbiome transplantation has recently been proposed for severe dysbiosis, with studies reporting improved donor engraftment, dysbiosis resolution, and live births in RPL cases [71]. Nutritional interventions, including prebiotics, omega‐3 fatty acids, and dietary fiber, can significantly influence microbiome composition and benefit women with RPL [72].

8. Study Limitations and Conflicting Evidence

Current research on the microbiome and RPL faces several limitations. Key challenges include the absence of standardized sampling protocols, variability across sequencing platforms, inconsistent thresholds for interpreting dysbiosis, and limited validation of therapeutic interventions in diverse populations. Small sample sizes, heterogeneous study cohorts, and differing analytical methods further restrict the generalizability of findings. Moreover, studies often report conflicting results regarding specific bacterial taxa, particularly Lactobacillus iners, which has been linked to negative outcomes in some cases but considered neutral in others. The temporal relationship between dysbiosis and pregnancy loss also remains unresolved: it is unclear whether dysbiosis contributes to RPL or arises because of underlying conditions that predispose individuals to pregnancy loss.

9. Clinical Implementation and Future Perspectives

Integrating microbiome analysis into RPL assessment requires standardization of collection protocols, analytical methods, and interpretation criteria [8, 22]. While new technologies, including comprehensive genetic analysis, cell‐level sequencing, and customized probiotics, have facilitated our understanding of the reproductive microbiome [7, 18, 68], technical limitations persist, including the risk of contamination with low‐biomass samples, challenges in sampling methodology, and variability in microbiome composition [11, 22] (Figure 4). Clinical evaluations must address significant individual variability, concerns about antibiotic resistance, and the safety of microbiome manipulation during pregnancy [13, 65] (Figure 4). Future research prioritizing mechanistic studies, longitudinal cohort studies, randomized controlled trials, and biomarker development is warranted to establish evidence‐based microbiome‐targeted interventions for the management of RPL.

FIGURE 4.

Clinical workflow for microbiome‐guided diagnosis and treatment of endometrial conditions. Clinical decision‐making pathway for diagnosing and treating endometrial conditions using microbiome testing. This involves sample collection, PCR or NGS‐based microbiome analysis, diagnostic evaluation, and therapeutic stratification into empiric or targeted interventions. This aims to guide clinicians toward personalized care based on microbial profiles and endometrial health status.

Funding

Grants from government or non‐government agencies did not support this study.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper

Authorship Statement

Conceptualization W.Y.A., W.B., data curation W.Y.A., W.B., I.S.; writing—original draft preparation, W.Y.A., S.F., writing—review and editing, W.Y.A., supervision, W.Y.A.; project administration, W.Y.A. and S.F.

Informed consent

No informed consent was required as it is a review article.