Advanced Technologies for Studying Microbiome-Female Reproductive Tract Interactions: Organoids, Organoids-on-a-Chip, and Beyond

Many microbes require specific structural and functional features to interact with their host epithelium, which could be impacted if the in vitro organoid model does not recreate relevant in vivo conditions. 51 As revealed in this section, this can involve mimicking an appropriate developmental stage, the mucus layer, and proper oxygen levels. Oxygen levels, which are not uniform across different bodily environments, play a crucial role in influencing microbial localization and various biological processes in the reproductive tract. 90 Low oxygen levels prevalent in reproductive tissues foster stem cell maintenance, while anaerobic or microaerophilic conditions favor the growth of specific reproductive tract microbes. 91 Conversely, changes in local oxygen levels during inflammation and disease may dramatically alter host–microbe dynamics. 92 However, replicating these conditions within organoid models is complex, as the standard oxygen concentration in organoid cultures (around 20%) contrasts sharply with the in vivo reality of less than 5% oxygen. 17 90 Creating stable and reproducible oxygen gradients across organoid cultures presents a technical challenge, but recent advances such as microfluidic organoid culture devices and oxygen-permeable scaffolds offer promising tools for maintaining controlled oxygen levels. 78 93 The human–microbial crosstalk module (HuMiX) represents a significant advancement in this field, allowing anaerobic bacteria to be maintained in an almost anoxic compartment. 94 The anoxic environment is created by perfusing an anoxic medium. Established in gastrointestinal host–microbe interaction systems, HuMiX consists of three parallel microfluidic chambers separated by semipermeable membranes with a modular design that facilitates disassembly and cell collection. Successfully co-cultured with anaerobic bacteria like Bacteroides caccae and Lactobacillus rhamnosus , as well as immune cells, the system is being further developed to become the immuno-HuMiX, allowing the coexistence of patient-derived microbiota, ECs, and immune cells, although the mucus-coated membrane currently prevents direct host–microbe contact. 94 Defining the optimal oxygen conditions for organoids and associated microbes is significant when developing a host–microbiome in vitro model. Modeling age-specific properties of the FRT epithelium is a key component for understanding fertility, particularly as aging profoundly affects the FRT's physiology. 95 EMOs derived from postmenopausal women exhibit altered morphology and reduced hormone responsiveness, highlighting the utility of organoids in modeling age-related properties of the FRT epithelium. 38 This also means that when generating adult stem cell-derived organoids, features like the age of the donor need to be accounted for, especially when conducting host–microbe studies, as age-specific properties have been shown to affect microbial communities. For instance, the ovulatory cycle's influence on microbial composition, with estrogen and progesterone causing changes in epithelial thickness and glycogen deposition, leads to different community state types with various Lactobacillus species. 91 Menopause results in a reduction in Lactobacilli , associated with higher follicle-stimulating hormone (FSH) levels and lower estrogen levels, linked to vaginal dryness and atrophy. 91 Moreover, postmenopausal estrogen deficit affects the vaginal microbiome, reducing Lactobacilli and correlating with decreased serum estrogen levels. 91 These insights into the age-specific properties of the FRT epithelium and their relationship with microbial interactions are vital for fertility studies. The ability to model these complex dynamics through organoids and other methods offers promising avenues for understanding and potentially addressing fertility challenges across various stages of a woman's life. The FRT is a dynamic system where the endometrium, ovaries, and cervix are influenced by hormonal changes that occur throughout the menstrual cycle, affecting cellular and molecular events, resident microbiota, and thereby host–microbe interactions. 95 These hormonal shifts, like rising estrogen levels, induce remarkable remodeling in the endometrium, affecting aspects like the proliferation of glands and the maturation of functional layers. 96 Turco et al used organoids to model how glandular EMOs respond to ovarian sex hormones, revealing specific hormonal effects on differentiation and showing the potential for studying the FRT microbiota's role in these processes. 38 Additionally, a microfluidic ovary–FT coculture device has demonstrated estrogen-mediated interorgan signaling. 97 Modeling the hormonal cycle in vitro is vital, as it would enable the study of dynamic changes such as pH, nutrient availability, and immune factors that influence host–microbe interactions throughout the cycle. The mucus layer in the FRT is vital for reproductive functions, with its properties varying due to hormonal changes, ensuring a conducive environment for fertilization and embryo gestation. 98 The cervico-vaginal tract acts as a barrier, balancing sperm passage, supporting beneficial bacteria, and warding off harmful pathogens. 99 100 Disturbances, like bacterial vaginosis, can cause health issues such as preterm birth and increased HIV-1 risk. 101 102 Hormonal cycles, especially estrogen and progesterone, influence mucus consistency and acidity. 103 While healthy mucus promotes Lactobacillus adherence and restricts pathogens, dysbiosis can impair fertility and lead to diseases like endometritis. 104 105 106 There is a need for human in vitro models that accurately represent mucus composition, structure, and function. Research has utilized various in vitro models, such as conventional cultures, transwell inserts, 3D-engineered constructs, and organoid models, to tackle these challenges. However, these models have limitations, and human organ-on-a-chip technology has been applied to overcome these challenges and model mucus biology more physiologically. 98 One notable advancement was made by Izadifar et al, who developed a two-channel cervical chip lined with primary human cervical ECs. 18 This innovation, along with the human vaginal chip, has provided insights into mucus physiology and pathophysiology. 89 A simplified alternative, the mucus chip, has also emerged, offering insights into mucus penetration for drug delivery. 107 Organ-on-a-chip technology's potential extends beyond modeling, facilitating simultaneous characterization of mucus biochemical, structural, and biophysical properties. 98 For future research, it would be useful to see if such FRT organoids-on-a-chip models can be made using organoid-derived cells or linearized organoids to have an accessible mucus layer. Then the properties between the differently constructed models' mucus layers could be assessed, both with microbial cultures and without. In summation, human organ-on-a-chip models represent an innovative platform for conducting mechanistic studies of mucus formation, functions, and underlying influences of tissue biology, holding great promise for studies on host–microbe interactions in the FRT. The mucus layer in the FRT regulates the microbial community, serving as a barrier that influences immune responses and reproductive health through intricate interactions. 108 109 110 111 Imbalances in reproductive tract microorganisms, metabolites, or immunity can disrupt this harmony, potentially leading to disease development. 112 Therefore, incorporating immune cells into organoid models enhances their ability to model infection, inflammation, and other disease processes driven by immune–microbe interactions. Incorporating immune cells into organoid structures entails methods like direct luminal injection, addition to culture medium, or leveraging organoids with inherent immune cells, the latter highlighted by Yu et al. 15 However, achieving uniform incorporation, phenotype maintenance, and accounting for variability in immune cell sources present challenges.

Organoid-derived technologies have significantly advanced the study of the FRT, filling the gap between traditional cell cultures and animal models. These advancements provide a basis for further refinement and standardization of these models. A defined research direction is crucial. Efforts should focus on standardizing FRT organoid–microbe co-cultures, considering variables such as donor age, hormonal cycles, and disease conditions. By addressing these elements, the creation of advanced models that simulate both aerobic and anaerobic environments becomes more achievable, leading to a closer representation of in vivo conditions suitable for both short- and long-term co-cultures. The subsequent step involves the development of interconnected multi-organoid systems, enhanced with microfluidic technologies, to reflect the cellular dynamics of the FRT more accurately. These models should aim to include vascularization, immune components, stromal elements, and other essential cell types. However, technical challenges, such as determining optimal flow rates and formulating compatible media, demand concentrated research efforts to ensure interactions with diverse cell types, microbes, and hormones. Investigating conditions like endometriosis using patient-specific organoid models is of paramount importance. Additionally, the merging of organoid technology with genomics, proteomics, and interdisciplinary research offers a broader perspective on FRT health. 40 Improvements in co-culture systems, biomaterials, and integrated screenings are promising pathways toward mimicking the in vivo microenvironment more closely. In conclusion, as organoid-based models continue to progress, their combination with microfluidic technologies, despite inherent challenges, stands to enhance our understanding of microbial roles in reproductive health, setting the stage for tailored therapeutic approaches in reproductive healthcare.

Established organoid models of the FRT include organoids of the endometrium, FT, ovaries, and cervix and have previously been summarized by Alzamil et al. 16 Endometrial organoids (EMOs) that form gland-like structures and respond to hormones enable the study of the menstrual cycle and infertility-related defects. 37 38 FT organoids, containing both secretory and ciliated cells, model oviduct physiology. 39 There are two main methods for FT organoids, the air–liquid interphase system and self-organizing organoids. 40 Cervical organoids effectively replicate both squamous and columnar epithelium, offering valuable insights into cervical biology. 41 42 These organoids, developed from both human and mouse tissues, rely on specific growth factor combinations ( Table 1 ) for their formation and long-term expansion, with distinct requirements for ecto- and endocervical organoids. 43 They serve as pivotal models for understanding cervical homeostasis, with their reactions to factors like Wnt growth mirroring in vivo dynamics, such as metaplasia at the squamocolumnar junction. Moreover, recent advancements have led to the creation of ovarian cancer organoids, which closely resemble the original tumors in histological and genomic features, offering potential for drug screening and understanding tumor dynamics. 43 44 Kopper et al demonstrated the development of organoids from human ovarian surface epithelium, especially from women predisposed to ovarian cancer due to BRCA1/2 mutations. 45 These ovarian surface epithelium organoids, mirroring features like FT organoids with KRT8+ markers and distinct folds, face growth and long-term maintenance challenges. On the other hand, murine ovarian surface epithelium organoids thrive and can be perpetually cultured under certain conditions. 45 These innovative models showcased ciliated and secretory cells, but optimization was needed to model ovulation. Recently, ovarian organoids, or “ovaroids,” containing oocyte progenitor cells and supporting granulosa cells have been generated from human-induced pluripotent stem cells (iPSCs). 46 These ovaroid cells model follicle development, oocyte maturation, and hormone secretion. Lastly, robust human vaginal tissue models are still in development. 43 Currently, mouse-derived organoids replicate the in vivo architecture of vaginal tissue, displaying a stratified squamous epithelium with TP63+ cells. The precise modulation of Wnt is crucial for vaginal epithelial behavior, making this model pivotal for exploring vaginal epithelium regeneration and stability. 47 The majority of FRT organoids are derived from adult stem cells located within epithelial tissue fragments isolated from surgical samples or biopsies. 16 The tissue is enzymatically digested, and the fragments containing stem cells are embedded in extracellular matrix gels and overlaid with defined media to promote the proliferation of stem cells and their self-organization into organoids. 39 48 The specific media formulations vary based on the source of the FRT tissue but typically contain niche factors like WNT activators like WNT3A, bone morphogenetic protein, transforming growth factor inhibitors like A83–01, and mitogens like epidermal growth factor (EGF), R-spondin 1 (RSPO1), and fibroblast growth factor 10 (FGF10) to support stem cell growth while preventing differentiation ( Table 1 ). 37

Choosing an accurate method for introducing microbes or microbial products to organoids is vital for effective in vitro modeling of host–microbe interactions within the FRT. 49 The type of microbes involved, compatibility with natural conditions like pH and oxygen levels, as well as study requirements such as duration, scalability, and throughput must be considered. While many studies focus primarily on bacterial abundance, it is important to mention that bacterial load, as for instance quantified by 16S qPCR, is believed to significantly contribute to the effects of dysbiosis on the FRT health, 50 which should be considered by choosing the right bacterial concentration when studying interactions in a co-culture. By evaluating these variables, researchers can select the organoid system that best aligns with their goals, ensuring consistency and a robust understanding of complex relationships within the FRT. Several options have been emerged, namely, suspension coculture, fragmentation of the organoids, micro-injection of the organoids, and organoid polarity reversal ( Fig. 1 ) which will be described later. Each model has its advantages and disadvantages of which an overview is given in Table 2 . 1 Integration techniques of microbes into female reproductive tract organoids. ( a ) Suspension culture : organoids are cultured in medium that allows free-floating conditions and enhances cell-to-cell interactions and nutrient absorption. Microbes are introduced into this medium, enabling interaction with the basal side of the organoid structure. ( b ) Microinjection : This technique employs a needle to inject a defined quantity of microbial suspension directly into the organoid's lumen, ensuring localized exposure of the apical side to the microbial agents and facilitating controlled studies of intraluminal microbial effects. ( c ) Fragmentation : Intact organoids are mechanically or enzymatically cleaved into smaller fragments, which are then replated and in the presence of microbial cultures both surrounding the organoid and within the organoid lumen. ( d ) Apical out organoids : The polarity of organoids is reversed to expose the apical surface, which typically lines the organoid lumen, to the external environment. This allows for the direct application of microbes onto the apical interface of the organoids. ( e ) 3D to 2D transition on transwell : Organoids are dissociated to either fragments or into single cells and seeded onto a porous membrane on a transwell. The cells grow into a confluent monolayer, serving as a model of the epithelial barrier. Microbes are added to the upper chamber to study their effects across the epithelial cell layer. ( f ) Organoid-on-a-chip : This method incorporates organoids into a microfluidic system designed to emulate the physical and biochemical aspects of their native tissue environment. Organoids are cultured within defined compartments, and microbes are introduced through microchannels, allowing for real-time observation of dynamic host–microbe interactions under controlled shear stress and fluidic conditions. Culturing organoids in a suspension culture is a widely utilized technique in host–microbe interaction studies. 51 This approach involves the culturing of organoids in a liquid medium to which specific bacteria, isolated bacterial products, or cell-free bacteria-conditioned culture medium can be added. 35 51 52 53 54 This approach presents challenges, such as the restriction of bacterial access to the apical side of the organoid or bacterial overgrowth. Yu et al utilized this method for inoculating patient-derived FT organoids with L. crispatus and Fannyhessea vaginae . 15 Results demonstrated significant differences in the expression of inflammatory genes in organoids cultured with either bacterial species. Similarly, Koster et al utilized the suspension technique to investigate the role of pathogenic bacteria in cervical mucosa coinfections using patient-derived ectocervical organoids emphasizing the potential of patient-derived ectocervical organoids as a tool for better understanding coinfections and their role in disease development. 14 Fragmentation is a technique that involves breaking down organoids into fragments and mixing them with bacteria or bacterial products before reseeding them into the extracellular matrix gel. 10 While this method facilitates bacterial interaction with both the apical and basal facets of epithelial cells, its physiological relevance is debated. Concerns arise from inconsistencies in the quantity of bacteria or bacterial products captured within each organoid and the potential for bacteria to engage with both the basal and apical surfaces. 10 51 Despite this, two exemplary studies have used this method to study the long-term effects of Chlamydia trachomatis infections in the FRT. 11 12 Kessler et al utilized human FT organoids to explore the enduring effects of C. trachomatis infections, a leading cause of tubal infertility. 11 Long-term infected cultures consistently expanded over numerous passages and molecular analyses at 9 months revealed lasting impacts of chronic C. trachomatis infection, including heightened stemness, altered epithelial renewal, and DNA hypermethylation, potentially predisposing to high-grade serous ovarian cancer (HGSOC). Bishop et al fragmented murine EMOs and infected them with mCherry-expressing C. trachomatis . 12 This method allowed the EMOs to undergo a full developmental cycle, offering novel short-term insights into C. trachomatis infections. These two studies underscore the efficacy of fragmentation and pave the way for a deeper exploration of the endometrium's interactions with diverse microbes using this technique. Microinjection is a technique that allows bacteria to contact the apical side of the epithelium while preserving the organoid's 3D structure as microbes are directly injected into organoid lumens. 55 Apical exposure is essential when studying pathogens like Salmonella enterica , which stimulate cytokine production in organoids only when applied to the apical side. 51 56 The injection of organoids has been extensively used in intestinal organoids and has been optimized using optimal injection volumes, fluorescent bacteria visualization, repeated microinjections, and high-throughput microinjection platforms. 55 Both commensal and pathogenic bacteria have been microinjected into organoids, with commensal bacteria maintaining species diversity for over 96 hours. 57 Repeated microinjections of genotoxic Escherichia coli led to mutational changes like colorectal cancer signatures, emphasizing the carcinogenic effect of this strain. 58 Microinjection offers advantages such as precise dosing of bacteria, repeated microinjections, hypoxic conditions, and longer experimental durations for studying organoid proliferation and epithelial cell subtypes. 51 58 However, it can cause structural damage to organoids, and the low oxygen in the organoid lumen makes replicating the true oxygen levels of specific FRT regions unfeasible. This approach is also susceptible to bacterial “spillage” into the basolateral compartment and is generally not ideal for high-throughput applications due to its high costs and technical challenges. 51 59 60 Despite these limitations, microinjection has proven to be a high-potential technique and is notably suited for brief analyses involving strict anaerobes due to low oxygen levels in the organoid lumen and it facilitates a direct interface between microbial and epithelial cells. 55 61 62 63 Microinjection has not been applied in many studies on the FRT. Two studies by Dolat and Valdivia utilized EMOs to investigate the interactions between Chlamydia and epithelial and immune cells in the upper genital tract. 13 The study revealed how C. trachomatis impacted the epithelial barrier and used co-cultured EMOs with mouse bone marrow–derived neutrophils to closely observe_immune cell recruitment. In subsequent research, they examined how C. trachomatis disrupts epithelial tight junctions by microinjecting EMO with specific bacterial strains. 64 The study revealed that C. trachomatis disrupts epithelial barriers by using the effector protein TepP to disassemble tight junctions early during infection. The injection of organoids offers a technical challenge, but when mastered can be especially useful when studying bacteria requiring low oxygen levels. A novel technique allowing for apical interaction with bacteria, or material products, has been developed by reversing the polarity of 3D intestinal organoids by first culturing them in a basement matrix dome, then dislodging and solubilizing the domes in PBS without Ca 2+ or Mg 2+ , centrifuging the organoid suspension, and finally resuspending the pelleted organoids in growth medium in a low-attachment tissue culture plate. 65 This method ensures proper epithelial barrier integrity and nutrient uptake without the need for microinjection, letting microbes interact directly with the organoid's outward-facing apical surface. For instance, Co et al revealed that invasive pathogens such as Salmonella typhimurium and Listeria monocytogenes exhibited distinct invasion strategies for polarized epithelium, 66 while enteropathogenic E. coli attached primarily to the apical side of mucin-secreting cells on inverted organoids. 67 Despite these advancements, the method has notable limitations, including the time required for reversal, increased cell death, and a tendency for inverted organoids to adhere to each other in the absence of an extracellular matrix gel. 66 However, Ahmad et al were able to use this technique to study the interaction of EMOs with murine blastocysts and E. coli . 68 Future research is needed to understand the impact of polarity inversion on apical-out organoids' phenotype, metabolism, and microbial response, providing insights into similarities and differences between this model and self-organized organoids. Once these fundamentals have been established, more intricate investigations can be conducted. Furthermore, a shared challenge with other techniques is that there is a risk of uncontrolled bacterial growth and toxins, upsetting the equilibrium that manages bacterial proliferation and toxin neutralization. This in turn impacts organoid integrity and function, 66 increasing unpredictability in maintaining balance over extended periods, thus underscoring the importance of precision in studies centered on steady host–microbe interactions. To date, this technique has yet to be established for studying microbiome-organoid interaction with FRT organoids. Transitioning organoids into 2D monolayers offers easier access to the apical side for introducing bacteria or their by-products, overcoming challenges posed by 3D cultures such as limited uniform access, complex cell–cell interactions, heterogeneity, and restricted high-throughput application potential, all while maintaining the inherent properties of the organoid system. 69 Organoids are linearized by fragmenting 3D structures into small cell clusters or individual cells, which are then plated onto extracellular matrix-coated surfaces to form a monolayer. 70 71 72 While sacrificing the 3D structure, 2D organoid monolayers have proven their utility for studying intestinal epithelial integrity upon exposure to microbes. 73 By employing transwells to segregate the apical and basal compartments, this technique can further refine EC (endothelial cell) differentiation and allow for the inclusion of additional host factors like mesenchymal cells or immune cells, more closely mimicking the in vivo environment of the FRT. 74 Furthermore, organoids on a transwell can be cultured with medium in both the apical and basal compartments or as an air–liquid interface culture. 74 The air–liquid interphase approach provides a physiologically relevant environment for certain cell types, exposing the apical side of the monolayer to air while the basal side stays submerged in media. 75 76 Expanding on this, Zhu et al developed a 3D air–liquid interphase culture with vaginal epithelium to study herpes simplex virus-2 (HSV-2) infections. This model not only demonstrates HSV-2 susceptibility but also offers valuable insights into infection mechanisms, potential drug targets, and therapeutic efficacy evaluations. 77 However, while 2D structures on transwell plates offer easier access to the apical side, a simplified structure, and suitability for high-throughput applications, they may also lead to the loss of intricate 3D interactions, potentially misrepresent the heterogeneity and maturation of organoids, and necessitate a higher number of ECs for initiation. 75 76 In the last decade, advances in tissue engineering, biofabrication, and microfluidics gave rise to “organ-on-a-chip” technology. 78 It is an engineered microfluidic 3D device which mimics the microarchitecture and functions of human tissues and organs. Each chip contains a polymer that has microfluidic channels lined by living tissue-specific cells and endothelial cells, under fluid flow conditions and mechanical forces to mimic organ movements. These microdevices can combine the different cell and tissue types making up human organs, thus representing an ideal approach to study organ function on the molecular and cellular level and mimic human-specific diseases. Each setup is different, depending on the organ's physiology. The integration of organoids with these microfluidic platforms has given birth to organoids-on-a-chip. 79 80 81 Growing organoids in a tightly regulated environment of micro-engineered system may enable the development of more realistic in vitro models not achievable with organoid approaches alone, allowing for precise spatiotemporal modulation of morphogens, nutrients, physiological forces, and vascularization by co-culturing with endothelial cells for vascularization. 78 82 83 84 This setup facilitates high-throughput drug testing, continuous organoid dynamics monitoring using embedded sensors. 78 85 86 The fluid flow in these platforms resembles in vivo conditions more closely, preventing microbial overgrowth, ensuring cell health, and promoting tissue-like organization, making them an interesting tool for studying microbiota–host interactions. 87 88 The potential of organ-on-a-chip technology in elucidating microbiota–host interactions is vividly demonstrated by Mahajan et al, who developed a “vagina-on-a-chip” model. 89 This model features a microfluidic culture system replicating the human vaginal mucosa, lined by hormone-sensitive primary vaginal epithelium interfaced with underlying stromal fibroblasts. Utilizing this innovative setup, they observed that coculture with the beneficial bacterium, L. crispatus , led to its successful engraftment and proliferation in the chip. This coculture also maintained an acid pH, produced both D- and L-lactate, and downregulated proinflammatory cytokines. In contrast, the presence of the non-optimal bacterium, G. vaginalis , elevated pH levels and increased the secretion of inflammatory cytokines, culminating in epithelial cell injury. Such precise control over oxygen and pH levels, demonstrated in this study, paves the way for future microbiome–host investigations involving different FRT tissues.