Organoid systems to study the human female reproductive tract and pregnancy.

How well do FRT organoids model the cellular heterogeneity of the tissue of origin? Are the different cell states across the menstrual cycle represented in the FRT organoid models? What are the signaling pathways and transcriptional networks regulating proliferation and differentiation of the organoids of the FRT? Can FRT organoids be used to generate more complex models that incorporate stromal and immune cell types? Can early developmental processes of pregnancy be modeled by co-culture of FRT organoids with embryos or trophoblast organoids? How well do FRT organoids model the cellular heterogeneity of the tissue of origin? Are the different cell states across the menstrual cycle represented in the FRT organoid models? What are the signaling pathways and transcriptional networks regulating proliferation and differentiation of the organoids of the FRT? Can FRT organoids be used to generate more complex models that incorporate stromal and immune cell types? Can early developmental processes of pregnancy be modeled by co-culture of FRT organoids with embryos or trophoblast organoids?

The efficient and coordinated function of the FRT is essential for reproduction and women’s wellbeing. Perturbations in these processes are the cause of a range of disorders from infertility to cancer. Organoids can be derived from healthy and pathological tissues of the FRT. Organoids of the reproductive tract faithfully recapitulate key morphological, functional, and molecular characteristics of their cells of origin. The organoid systems provide an essential tool to study the physiology and disease of the FRT. The efficient and coordinated function of the FRT is essential for reproduction and women’s wellbeing. Perturbations in these processes are the cause of a range of disorders from infertility to cancer. Organoids can be derived from healthy and pathological tissues of the FRT. Organoids of the reproductive tract faithfully recapitulate key morphological, functional, and molecular characteristics of their cells of origin. The organoid systems provide an essential tool to study the physiology and disease of the FRT.

Organoids can be established from pathological samples and provide powerful tools to study diseases ex vivo although the culture conditions often need optimizing [ 69 , 78 , 95 , 102 – 111 ] (Table  4 ). A common benign condition is endometriosis where ectopic foci of the endometrium containing both glands and stroma are found in the peritoneal cavity, ovary, and cervix. Active WNT and EGF, with inhibition of BMP signaling, are necessary for efficient growth of organoids from these lesions. Organoids from ectopic sites of endometriosis express genes linked to stemness ( SOX9, LGR6 ) and metalloproteinase function ( MMP2 ), and show luminal invasion, a common characteristic of endometriosis. Somatic mutations in genes associated with endometrial carcinoma ( CTCF, EP300, ZNF471 ) were found in organoids from late-stage endometriotic lesions. When engrafted into mice, they give rise to ESR1 + and PR + lesions. Using the same medium composition for endometriosis, organoids can also be established from endometrial hyperplasia. This is a condition usually found in anovulatory women exposed to estrogen without progesterone and is characterised by an abnormally thick endometrium. Organoids from endometrial hyperplasia  display high proliferative capacity and similar molecular characteristics as the primary tissue, including absence of TP53 expression. Mutations in mismatch DNA repair genes ( MSH2, MSH6 ) were observed in organoids derived from women with Lynch syndrome [ 108 ]. Table 4 Organoids derived from pathological tissues of the human FRT. Tissue Pathology Applications Medium components a Reference Ovaries Ovarian carcinoma Drug screening (multidrug assay) b F GF, E GF, I GF-I, β- m ercaptoethanol, H eparin, H ydrocortisone, I nsulin Y -27632, β- E stradiol [ 102 ] Prediction of DNA repair in response to inhibitors R SPO1, No ggin, N icotinamide, E GF, F GF-10, F GF2, P rostaglandin E2, A 83–01, S B202190 [ 103 ] Characterization, Gene editing, Drug screening, In vivo drug sensitivity R SPO1, W NT CM d , N oggin, N icotinamide, E GF, F GF-10, A 83-01, H eregulin-β, Y -27632, F orskolin, H ydrocortisone, β- E stradiol [ 69 ] Characterization, Drug sensitivity R SPO1, N oggin, E GF, Y- 27632, J agged-1 [ 104 ] Multi-drug screening P rEGMTM or M ammoCult e [ 105 ] Characterization, Optimization of culture conditions N icotinamide, E GF, S B431542, Y -27632, B MP2 [ 106 ] Fallopian tube Chlamydia infection Characterization, Investigation of chronic infection R SPO1, W nt3A, N oggin, N icotinamide, E GF, F GF-10, S B431542, Y -27632 [ 107 ] Endometrium Endometriosis Characterization, Analysis of ion channels R SPO1, N oggin, N icotinamide, E GF, b F GF, F GF-10, A 83-01, S B202190 (low), I TS, β- E stradiol b , Y-27632 c [ 108 ] Endometrial hyperplasia Lynch syndrome Endometrial carcinoma Evaluation of apoptotic effects of verteporfin E GF, b F GF, I nsulin, B SA [ 109 ] Evaluation of growth inhibitory effects of drugs N icotinamide, A 83-01, S B202190, Y -27632, β- E stradiol [ 110 ] Characterization R SPO1, N oggin, N icotinamide, E GF, F GF10, H GF, A 83-01 [ 78 ] High-throuput multidrug screening F BIM002 medium [ 111 ] Characterization R SPO1, N oggin, E GF, Y -27632, Jagged-1 [ 104 ] Characterization, Drug screening, Analysis of ion channels R SPO1 (low), N oggin, N icotinamide (high), E GF, I GF-1, H GF, A 83-01, S B202190, β E stradiol, Y-27632 c [ 108 ] Cervix Cervical clear cell carcinoma Characterization, Drug screening R SPO1, N oggin, E GF, Y -27632, Jagged-1 [ 115 ] Organoid models of pathologies of the human FRT are shown together with their applications. The medium composition with growth factors and inhibitors are shown and the basal medium components (e.g., DMEM/F12, N2, B27, N-acetyl cysteine, Hepes, and antibiotics) are not listed. RSPO1 R-spondin 1, EGF Epidermal Growth Factor, FGF fibroblast growth factor, HGF hepatocyte growth factor, IGF-1 Insulin-like Growth Factor-1, ITS Insulin Transferrin Selenium, A83-01 TGFβ receptor inhibitor, SB431542 TGFβ signaling inhibitor, SB202190 p38 MAPK inhibitor, Y-27632 ROCK signaling Inhibitor, BSA bovine serum albumin. a Excluding basal medium components. b Only for organoid expansion. c Only for organoid formation or dissociation at passaging. d WNT-conditioned medium was used only for some tumor organoid lines. e Serum-free commercial media (PrEGMTM Prostate Epithelial Cell Growth Medium, MammoCult; for culture of mammospheres from normal human primary breast tissues and tumorspheres from human breast cancer cell lines. Organoids derived from pathological tissues of the human FRT. Organoid models of pathologies of the human FRT are shown together with their applications. The medium composition with growth factors and inhibitors are shown and the basal medium components (e.g., DMEM/F12, N2, B27, N-acetyl cysteine, Hepes, and antibiotics) are not listed. RSPO1 R-spondin 1, EGF Epidermal Growth Factor, FGF fibroblast growth factor, HGF hepatocyte growth factor, IGF-1 Insulin-like Growth Factor-1, ITS Insulin Transferrin Selenium, A83-01 TGFβ receptor inhibitor, SB431542 TGFβ signaling inhibitor, SB202190 p38 MAPK inhibitor, Y-27632 ROCK signaling Inhibitor, BSA bovine serum albumin. a Excluding basal medium components. b Only for organoid expansion. c Only for organoid formation or dissociation at passaging. d WNT-conditioned medium was used only for some tumor organoid lines. e Serum-free commercial media (PrEGMTM Prostate Epithelial Cell Growth Medium, MammoCult; for culture of mammospheres from normal human primary breast tissues and tumorspheres from human breast cancer cell lines. Several organoid models have now been derived from carcinomas arising in the FRT (Table  4 ). Ovarian carcinomas are heterogeneous with high grade serous ovarian carcinoma (HGSOC) being the deadliest and most common type [ 112 ]. It seems that HGSOC originates from the epithelium of the fimbriae of the fallopian tube [ 113 ], although others have proposed the source is the OSE [ 114 ]. 3D cultures from solid tumors, ascitic, and pleural fluid of patients with ovarian carcinoma were generated [ 103 ]. Whilst these cultures displayed clonogenic capacity and morphological and molecular similarity to the primary tumors, they could only be expanded short-term. In another study, a biobank of organoids from pre-malignant and malignant ovarian neoplasms was established [ 69 ]. These organoids recapitulate morphological (nuclear and cellular atypia), phenotypic (PAX8 + , TP53 + ), and genomic features (mutations in KRAS, BRAF, cell cycle genes, and TP53), capture tumor heterogeneity and can be expanded long-term. However, these culture conditions are still suboptimal and HGSOC organoids grow slowly. Growth and long-term expansion has now been achieved by using media with low WNT and active BMP signaling to maintain stem cells in the cultures (Table  4 ) [ 106 ]. These conditions are also required for the stable growth of another model of HGSOC that were derived by the stable triple knockdown of TP53 , PTEN , and RB in fallopian tube organoids [ 106 ]. Similarly, organoids can be derived from different stages of endometrial neoplasms that capture the phenotypic and genetic heterogeneity (Table  4 ). Low WNT but high p38 MAPK signaling, both associated with cell proliferation and differentiation, together with high concentration of β-estradiol, addition of growth factors (IGF1, HGF) and lipids enhance the expandability of organoids derived from endometrial carcinomas [ 108 ]. Organoids from low-grade endometrial carcinomas bear mutations in tumor suppressor genes ( PTEN, CTCF, and ARID1A ) and the β-catenin coding gene ( CTNNB1 ), resulting in continuous activation of the WNT pathway. In contrast, organoids from high-grade endometrial carcinomas were characterized by downregulation of glandular markers ( ESR1, FOXA2 ) but upregulation of EMT-associated genes ( CXCR4, TWIST1, ZEB1, and CDH2 ) [ 108 ]. These organoids can be orthotopically engrafted into murine uterine horns where the histological and molecular features of the original lesion are retained with the potential to metastasize [ 108 ]. Organoids derived from clear cell carcinoma, a rare tumor of the cervix could be propagated for more than 6 months, retained 2 out of 3 mutations ( MLH1 and TFE3) detected in the original tumor and typical markers, HNF1‐β, TP53, and Ki‐67 [ 115 ] (Table  4 ). Their appearance was similar to the original tumor with atypical cells with clear cytoplasm. No organoid models of squamous cervical carcinoma have been reported. Patient-derived organoids from pathologies of the FRT can be frozen and thawed allowing generation of extensive biobanks, which can be used for drug sensitivity screening and ultimately for personalized medicine [ 69 , 102 , 104 , 105 , 108 ]. When treated with platinum/taxane, drugs commonly used for treating ovarian cancer, different organoid lines showed differential drug responses. Sensitive and resistant organoids correlated with the grade of the tumor (high or low) and the degree of chemoresistance previously noted in the patients [ 69 ]. Drug responses have also been examined in vivo in mice xenografted with organoids of ovarian carcinomas [ 69 ]. When the mice were treated with gemcitabine, a nucleoside analog commonly used for the treatment of HGSOC, proliferation, and invasion of the tumors was restricted. Similarly, patient-specific responses were also observed in organoids derived from endometrial carcinoma treated with standard chemotherapeutic compounds [ 108 , 110 , 111 ]. The resistance of the organoids to cisplatin and paclitaxel echoed the patients’ clinical response to treatment [ 111 ]. Another report described the induction of apoptosis in organoids from endometrial carcinoma in response to verteporfin, a drug which inhibits the HIPPO pathway [ 109 ]. Similarly, napabucasin, an inhibitor of STAT3 signaling hampered their growth [ 110 ]. This variability in drug response may be due to the lack of stromal or immune cells in these organoid models [ 116 , 117 ], which could be resolved with the development of co-culture systems.

Throughout adult reproductive life, the FRT undergoes constant remodeling under the influence of pituitary and ovarian hormones and, if pregnancy occurs, it goes through dramatic changes driven by placental hormones. Disruption of all these complex, exquisitely controlled processes results in a diverse range of pathologies that together affect a large number of women worldwide. Although much is known about hormonal changes occurring in the FRT, it is mostly descriptive without detailed molecular and cellular information. Now available are essential experimental organoid models of the FRT that recapitulate the original tissues (healthy or pathological). Although organoid cultures are more labor intensive and costly compared with standard 2D culture, they can be set-up with relative ease allowing wide-spread use. There are still many questions to be answered regarding the FRT organoids. Efforts are now being made to define the common issues affecting all organoids systems such as reproducibility, standardization, and diligence validation [ 141 ]. This is an exciting time for reproductive research as recent progress paves the way for opportunities to improve women’s wellbeing and reproductive health.

Combining techniques to engineer genomes of organoids from the FRT open up new possibilities to study its physiology and disease (Fig.  6a ). There is still much to learn about epithelial regeneration, maintenance, and differentiation of the FRT and it is evident that CRISPR/Cas9 gene editing technology will help address many outstanding questions. It allows site-specific targeting to disrupt or modify a genetic locus of interest. For example, transcription factors involved in proliferation and differentiation of the epithelial cells of the FRT can be targeted to study their functions. It can be used for creating reporter lines and performing lineage tracing experiments to identify progenitor populations of the different regions of the FRT. In a forward genetics approach, specific mutations can be introduced to study the etiology of endometrial and cervical carcinomas as previously done for targeting genes involved in ovarian cancer using fallopian tube organoids [ 69 ]. Fig. 6 Applications of tissue-derived organoids of the female reproductive tract and placenta. a Organoid systems can be used for studying the physiology and pathologies of the FRT. They can be used as. tools for testing drug responses and drug development. Gene function can be assesed by CRISPR/Cas9 based genetic engineering of organoids. b The possibility to combine organoids with different cell types as well as pathogens will allow studies on their interactions. Bioengineering methods may allow the generation of more complex tissue-like models that include non-epithelial populations such as fibroblasts, immune and endothelial cells. The interactions between two different tissues can also be studied by co-culture of organoids, which is of particular relevance for maternal–fetal crosstalk using placental (trophoblast) and endometrial organoids. a Organoid systems can be used for studying the physiology and pathologies of the FRT. They can be used as. tools for testing drug responses and drug development. Gene function can be assesed by CRISPR/Cas9 based genetic engineering of organoids. b The possibility to combine organoids with different cell types as well as pathogens will allow studies on their interactions. Bioengineering methods may allow the generation of more complex tissue-like models that include non-epithelial populations such as fibroblasts, immune and endothelial cells. The interactions between two different tissues can also be studied by co-culture of organoids, which is of particular relevance for maternal–fetal crosstalk using placental (trophoblast) and endometrial organoids. One of the exciting prospects of the generation of organoids from the different regions of the FRT is the possibility of personalized medicine (Fig.  6a ). This is especially relevant for studying infertility. Even though all patients undergoing in vitro fertilization (IVF) have the same hormonal treatment, the cause of the infertility is unexplained in ~30% of cases [ 118 ]. Organoids could be generated from endometrial biopsies from women undergoing IVF and exposed to these hormonal regimes to investigate whether differential responses may be one of the possible causes. Treatment regimens could then be tailored to each patient. A similar approach could be used for treating endometriosis where the eutopic endometrium is often progesterone resistant and patients have fertility issues [ 119 ]. Further optimization of organoid models of the healthy ovarian epithelium could prove useful in treating suboptimal infertility caused by anovulation that occurs in women with polycystic ovary syndrome. Ovulation induction is one of the most common therapeutic approaches. This comes with risks of ovarian hyperstimulation syndrome [ 120 ], causing patients a range of symptoms (abdominal pain, vomiting, fluid accumulation in the abdomen, and lungs) that may require hospitalization. In vitro maturation of eggs, using ovarian organoids would be a less invasive and risky approach for assisted reproductive technology. Tissue-derived organoids of the FRT include only the epithelial compartment and thus do not fully  reflect the cellular complexity of the native tissues. For the study of cellular interactions, multicellular organoid co-culture systems are necessary (Fig.  6b ). The interdependence of endometrial epithelial cells with stromal cells in the endometrium has been studied extensively using animal models but this is difficult to model in humans [ 121 , 122 ]. Stromal cells respond to estrogen and progesterone by producing prolactin and IGFBP1, which act on the glands to stimulate secretions [ 123 ]. In an organotypic approach, glandular epithelial cells embedded in Matrigel were cultured on a monolayer of stromal cells to investigate the effects of steroid hormones and anticancer drugs on the epithelial compartment [ 124 ]. Alternative methods for epithelial-stromal co-cultures utilize scaffolds. This also cirumvents the use of Matrigel which is not chemically defined and has batch-to-batch variation [ 125 ]. Recently, a porous collagen-based co-culture model of the epithelial and stromal cells of the endometrium was developed with both cell types being functionally responsive to hormones [ 126 ]. In future, the architecture of these scaffolds could be manipulated to allow the formation of gland-like structures that closely resemble the native tissue. Careful comparison with information about the in vivo environment is essential to prove the validity of the models [ 127 ], and to identify the characteristics of the niche that target epithelial populations [ 128 ]. Although these systems show promise in understanding the endometrium as a whole, there are still issues to resolve like finding media appropriate for all cell types. Immune cells play a major role in the homeostasis and function of the FRT. In the endometrium, progesterone upregulates IL-15 that is trans-presented by stromal cells to stimulate the proliferation and differentiation of uterine natural killer cells [ 129 ]. How these hormonally-regulated, distinctive, uterine lymphocytes play a role in epithelial regulation and function has been challenging to study. Co-culture methods that combine epithelial organoids with immune cells provide insights into normal intestinal homeostasis and diseases including gastric, pancreatic, colorectal, lung, and breast neoplasms [ 130 ]. Similar co-culture systems of organoids of FRT with immune cells will clearly be an important advance (Fig. 6b ). They will also be important in understanding behavior of tumors of the FRT where the microenvironment plays a crucial role in the development and progression of the disease [ 131 ]. A further use of multicellular culture models of the FRT would be to explore early developmental events in humans that are impossible to study in vivo (Fig.  6b ). Though such studies need to be scrutinized appropriately and ethical considerations must be taken into account [ 132 ]. An artificial endometrium could be used to model implantation in a dish [ 133 , 134 ]. Previous attempts to study the attachment of the blastocyst or trophoblast onto the endometrium [ 45 , 46 , 48 , 135 ] used epithelial monolayers superimposed on stromal cells, thus not recapitulating the in vivo 3D glandular structure. An important application of trophoblast organoids that differentiate to invading EVT, will be to study molecular and functional interactions with decidual cells (uterine NK, macrophages, epithelial, and stromal cells) in vitro. Organoids are a practical model to study infectious diseases [ 65 ]. Tubal infections caused by sexually transmitted diseases, misuse of intrauterine contraceptive devices, or post-miscarriage, can lead to tubal fibrosis, loss of patency, and infertility [ 136 ]. Such infections have also been linked to cancer [ 137 ]. The most common pathogen is Chlamydia trachomatis . A microarray analysis of infected organoids during the acute phase of a Chlamydia Ctr serovar D infection shows prolonged activation of leukemia inhibitory factor signaling that could play a role in maintaining stem cell identity (Table 4 ) [ 107 ]. The robust activation of paracrine networks controlling not only cell growth and proliferation, but also differentiation and cell fate, suggests that Ctr infection has pervasive long-term consequences on the epithelium. Pathogens of the lower reproductive tract including vaginal bacterial species (lactobacilli and vaginosis associated bacteria) can ascend to the endometrial cavity [ 138 ]. Infection with Neisseria gonorrhoeae of 3D epithelial cultures derived from endometrial adenocarcinoma resulted in upregulation of proinflammatory mediators and morphological changes to the host cells [ 139 ]. Endometrial organoids will be a valuable model to investigate chronic endometritis commonly caused by Chlamydia trachomatis , Neisseria gonorrhoeae, and Trichomonas vaginalis [ 140 ]. Co-culture of immune cells with the organoids will increase understanding of the pathogenesis of endometritis (Fig.  6b ). The major disease affecting the cervix is dysplasia and subsequent carcinoma caused by high-risk human papilloma virus (HR-HPV) [ 90 ]. Modeling the normal metaplastic change from glandular to squamous epithelium and the response of the epithelium to HPV infection with progression to carcinoma will provide information about the dynamics of epithelial renewal. In addition, cervical organoids can offer a system to study sexually transmitted diseases ( Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and herpes simplex virus).

The female reproductive tract (FRT) develops during fetal and early postnatal life [ 1 ]. It is derived principally from the Müllerian ducts (also referred to as the paramesonephric ducts) that develop from the intermediate mesoderm of the urogenital ridge on either side of the midline during 6–9.5 gestational weeks in humans [ 2 ]. Initially, this structure is present in both sexes, but the secretion of anti-Müllerian hormone results in regression of the Müllerian ducts in males [ 3 ]. In contrast, in females, they differentiate to develop into the fallopian tubes, uterus, cervix, and upper vagina. The ovaries are derived from the genital ridge which forms on the medial side of the urogenital ridge. The proper development and functioning of the FRT are essential for it to fulfill its ultimate goal of reproduction that includes the physiological processes of oocyte maturation, fertilization, implantation, fetal growth, and parturition. Disorders of FRT that include carcinomas of the cervix and endometrium, endometriosis, infertility, and heavy menstrual bleeding are alarmingly common and are a source of major suffering [ 4 ]. Effective treatments are still lacking for many of these conditions. Furthermore, factors such as obesity and delaying the age of reproduction have led to an increase in prevalence in infertility and uterine cancers [ 5 , 6 ]. Thus, understanding the biology of the FRT is becoming of increasing importance. Neither the biology of the normal human FRT nor the etiology of most of its disorders is clearly understood because they have been difficult to study ex vivo . The most commonly used in vitro models include primary cells, cancer cell lines, tissue explants, and organotypic cultures [ 7 – 59 ] (Table  1 ). However, they have limitations. Primary cells from tissue biopsies cannot be propagated indefinitely and lose their epithelial phenotype. Available cell lines are often derived from cancers or are immortalized and do not represent cells in their normal physiological state. Although cell lines derived from endometrial (e.g., Ishikawa, ECC-1) [ 26 , 60 ], cervical (e.g. HeLa [ 30 ], SiHa [ 31 ], C33a [ 32 ]), and ovarian carcinomas [ 7 , 61 , 62 ] have been essential tools to study these tumors, they do not maintain the original cellular heterogeneity due to selection of cells with proliferative advantage. Furthermore, culturing cells in two-dimensional (2D) monolayers deprives them of the three-dimensional (3D) environmental stimuli from the surrounding matrix that play a major role in cellular behavior [ 63 ]. The study of the physiology of pregnancy in humans has also been a major challenge because, compared with other organs, the reproductive tract and its association with the placenta are the most evolutionary diverse across species. Many of the human-specific features are modeled only to a limited extent in animal models and available in vitro tools. Thus, long-term physiologically relevant models that recapitulate epithelial architecture, cellular heterogeneity and functionality of the different regions of the human FRT are essential. Table 1 In vitro models of the human FRT used before the establishment of organoid systems. Cell culture system Tissue Model Reference a 2D Ovaries Tissue-derived ovarian surface epithelial cultures [ 7 – 9 ] Immortalized ovarian surface epithelial cultures [ 10 – 12 ] Fallopian tubes Tissue-derived epithelial cultures [ 13 ] Immortalized epithelial cultures [ 14 – 16 ] Endometrium Tissue-derived epithelial cultures [ 17 – 19 ] Tissue-derived stromal cultures [ 18 , 20 ] Tissue-derived endothelial cultures [ 21 – 23 ] iPSC-derived stromal cultures [ 24 ] Carcinoma-derived cell lines (HEC-1, Ishikawa, RL95–2, St-1b, ECC-1) [ 25 – 29 ] Cervix Tissue-derived epithelial cultures [ 30 – 32 , 34 , 35 ] HPV16-immortalized epithelial cell line [ 36 ] Carcinoma-derived cell lines (HeLa,SiHa,C33a,CaSki,ME-180) [ 33 , 37 ] 3D Ovaries Tissue-derived epithelial spheroid cultures [ 38 , 39 ] Fallopian tubes Tissue-derived epithelial spheroid cultures [ 40 ] iPSC-derived epithelial cell 3D cultures [ 41 ] Endometrium Tissue-derived epithelial cell 3D cultures [ 42 ] Organotypic cultures from endometrial tissue [ 43 – 46 ] Mesenchymal-derived epithelial-like cell 3D cultures [ 47 ] Spheroids derived from endometrial adenocarcinoma cell lines [ 48 ] Spheroids of mesenchymal stem cells derived from menstrual blood/endometrial fragments [ 49 ] Cervix Organotypic cultures from primary cervical epithelial cells [ 50 , 51 ] Organotypic cultures from HPV16-immortalized cells [ 50 ] Tissue explants Ovaries Ovarian tissue explants [ 52 ] Fallopian tubes Fimbria explants [ 53 , 54 ] Endometrium Non-pregnant endometrium explants [ 55 , 56 ] 1st trimester decidua parietalis explants [ 57 , 58 ] Cervix Cervical explants [ 59 ] A summary of the different in vitro tools available for studying the biology of human FRT (excluding the recently derived organoid models). The models have been grouped according to the culture type: 2D monolayer cultures, 3D models and tissue explants. A few representative references are cited per type of model. a Reprentative references of each model. In vitro models of the human FRT used before the establishment of organoid systems. A summary of the different in vitro tools available for studying the biology of human FRT (excluding the recently derived organoid models). The models have been grouped according to the culture type: 2D monolayer cultures, 3D models and tissue explants. A few representative references are cited per type of model. a Reprentative references of each model. Culturing cells in 3D is a technique that has been employed for decades using a variety of methods, but these were not chemically defined nor standardized (Table  1 ). In 2009, Sato and Clevers described a 3D culture system that has led to the systematic generation of organoids from many different organs [ 64 ]. Organoids are 3D cellular structures that retain functional and morphological features of tissues [ 65 ]. They can be derived from tissues or pluripotent stem cells. For the generation of organoids from tissues, primary cells are embedded into a hydrogel containing extracellular proteins, usually the commercially available Matrigel isolated from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, which acts as its basement membrane. Cells are cultured in a medium that recapitulates signals from the specific niche for each tissue. The organoid technology has several major advantages: long-term propagation of primary cells; recapitulation of the molecular, and functional characteristics of the tissue; genetic stability over time; freezing/thawing allowing bio-banking; and the ability to manipulate experimentally with a range of approaches [ 65 ]. Organoid technology is now transforming the way we study the FRT in physiology and disease. Here, we provide a general overview of the female reproductive system and describe the organoid models available to study its biology in health (including pregnancy) and disease (Fig.  1 ). We focus on organoids derived from human adult tissues. We include trophoblast organoids as, although the placenta is an organ of fetal origin and is not part of the FRT, it is closely associated as a functional unit with the uterine lining, the decidua, during pregnancy. Fig. 1 Tissue-derived organoids of the human FRT and placenta. a Types of organoids derived from the human FRT using tissue samples (normal and pathological) from non-pregnant women: ovaries, fallopian tubes, endometrium, and cervix. Organoids can also be derived from disorders of the FRT such as endometriosis and cancer. The different organoid systems show specific features that recapitulate the epithelial organization of their tissue of origin. b Organoid systems that have been derived from the pregnant endometrial lining (decidua) and the first-trimester placenta (fetal origin). a Types of organoids derived from the human FRT using tissue samples (normal and pathological) from non-pregnant women: ovaries, fallopian tubes, endometrium, and cervix. Organoids can also be derived from disorders of the FRT such as endometriosis and cancer. The different organoid systems show specific features that recapitulate the epithelial organization of their tissue of origin. b Organoid systems that have been derived from the pregnant endometrial lining (decidua) and the first-trimester placenta (fetal origin).