Development and Applications of Organoids in Gynecological Diseases.

Drug development is inherently high-risk with a high failure rate. Vaidyanathan [ 48 ] conducted a statistical analysis of 144 phase II clinical trials from 2008 to 2010 and 166 phase III clinical trials from 2007 to 2010, revealing a 75% failure rate. Achieving the correct balance of clinical dose, efficacy, and toxicity is critical for improving clinical drug development. Utilizing organoids in the early stages of drug development can aid in screening drugs that are effective for most patients or those with specific mutations. Organoids can be sequenced using whole-genome or whole-exome techniques derived from patients’ diseased tissues to identify key gene mutations for targeted drug prediction. They can also simulate the vivo microenvironment to faithfully replicate the original characteristics of multifocal lesions or be directly transplanted into nude mice to observe in vivo growth dynamics. Both methods enable the analysis of cellular activity and survival rates following treatment with different drugs at varying concentrations. Multiple tests can be conducted in parallel to screen for effective drugs. The organoid platform can simultaneously assess both tumor and healthy organoids, rapidly identifying drug side effects on healthy tissues, which facilitates early identification of drug toxicity, efficacy evaluation, and optimization. Organoids have emerged as a promising tool for predicting drug sensitivity and anticipating a patient’s clinical response prior to treatment. In 2019, Boretto’s team [ 39 ] and Kopper’s team [ 33 ] successfully established endometrial cancer and ovarian cancer organoids from living tissue samples, respectively. These organoids effectively recapitulated the histological and heterogeneous features of the primary diseases while preserving key genetic and molecular characteristics. Upon testing with conventional chemotherapy agents, the organoids exhibited patient-specific disease responses, underscoring their potential as preclinical models. de Witte et al. [ 49 ] developed multiple patient-derived organoids (PDOs) from primary and diffuse foci of different patients, as well as from the same patient, revealing a strong correlation between tumor heterogeneity and drug sensitivity. These PDOs were exposed to various chemotherapy drugs and exhibited drug responses in vitro that closely mirrored clinical outcomes. Bi et al. [ 50 ] tested the drug sensitivity of eight endometrial cancer organoids to paclitaxel, carboplatin, cisplatin, bevacizumab, gemcitabine, and topotecan. Furthermore, they utilized PDO models of advanced high-grade serous endometrial carcinoma treated with neoadjuvant trastuzumab to successfully predict trastuzumab resistance in patients undergoing postoperative chemotherapy. Following the development of trastuzumab resistance, they identified a new chemotherapy regimen, with the combination of carboplatin, paclitaxel, and bevacizumab being the most effective, providing a basis for clinical diagnosis and treatment. The PDO model not only predicts drug resistance but also facilitates the screening of new chemotherapy regimens and the identification of alternative treatment options currently employed in second-line settings. Seol et al. [ 51 ] assessed the sensitivity of four cervical cancer organoid subtypes—squamous cell carcinoma (SqCa), adenocarcinoma (AdCa), adenocarcinoma follicularis (VGA), and neuroendocrine cervical cancer (NECC)—to chemotherapy regimens including paclitaxel, carboplatin, cisplatin, Ly290042 , and rapamycin. Additionally, they screened seven compounds that significantly inhibited organoid activity in cervical cancer, demonstrating the utility of PDOs in evaluating drug response and resistance to targeted therapies. Given that radiation therapy is a primary treatment option for cervical cancer, they conducted the first radiation-sensitivity test on organoids. They found that SqCa and VGA were sensitive to radiation therapy, while AdCa and NECC were resistant, consistent with clinical observations. These findings suggest the potential of cervical organoids as a tool for radiation therapy as well. Cancer drug resistance refers to the phenomenon where tumor cells develop resistance to therapeutic drugs, leading to diminished or even ineffective treatment outcomes [ 52 – 54 ]. Investigating the mechanisms underlying tumor resistance can aid in the development of new drugs or therapies that overcome or circumvent this resistance, thereby enhancing the overall effectiveness of cancer treatment. Gorski et al. [ 55 ] cultured six HGSOC organoids and used varying doses of carboplatin to predict patient resistance and sensitivity to platinum. Their comparison revealed that TEME178B might be a previously unreported gene associated with chemotherapy resistance. Similarly, Sun et al. [ 56 ] identified Aurora A as a drug resistance target through RNA sequencing analysis. Wang et al. [ 57 ]simulated the development of drug resistance by incrementally increasing platinum concentration and identified fibrinogen 1 as a target for chemoresistance. Understanding tumor resistance mechanisms through organoid models enables the discovery of novel resistance targets and the development of new targeted treatment strategies. Kiyohara et al. [ 58 ] screened 79 molecular-targeted drugs using cancer tissue-originated spheroids derived from grade III endometrioid and serous adenocarcinoma tissues, focusing on two drugs, everolimus and YM155. Their study found that growth inhibition in everolimus-sensitive endometrial carcinoma spheroids was due to the inhibition of proliferation rather than the induction of cell death. The phosphorylation of downstream molecules of mTORC1, particularly S6, was significantly inhibited by everolimus treatment, while the phosphorylation of 4EBP1 was moderately inhibited, indicating that it cannot be used as a definitive biomarker to predict drug efficacy. YM155 was found to induce cell death, and experimental results confirmed that histological type is related to YM155 sensitivity. Endometrial carcinoma spheroids of non-endometrioid adenocarcinoma were all sensitive to YM155, but this was shown to result from autophagic cell death via a non-classical pathway, providing new therapeutic insights. Additionally, the feasibility of using PDO culture of endometrial cancer for drug screening of individual patient tumors was demonstrated. In 2021, Berg et al. [ 59 ] established five independent PDOs from a case of endometrial clear cell carcinoma, but did not extensively study tumor heterogeneity. In contrast, Maru et al. [ 60 ] generated three PDOs from different lesions of a stage IVB uterine carcinosarcoma case in 2024, offering a new model for studying tumor heterogeneity. For the first time, their diversity was validated through comprehensive analysis, contributing to a deeper understanding of advanced endometrial cancer complexity. The patient subsequently received a combination of adjuvant chemotherapy and molecular-targeted therapy, the tumor exhibited disease progression. The residual tumor cells are likely to develop resistance to standard EC drugs, with spatial tumor diversity potentially underlying the high-risk basis for acquiring a resistant phenotype. To identify potentially effective inhibitors for this patient, 361 compounds were screened across three PDOs using high-throughput techniques, yielding four drug candidates: staurosporin, ouabain, MG-132, and bortezomib. All candidate compounds reduced PDO activity in a dose-dependent manner, with bortezomib and staurosporine showing the most potent inhibitory effects. The application of organoids in drug screening demonstrates significant potential in the development of personalized treatment strategies. These strategies can predict patient responses to specific drugs, drive new drug development, personalize treatment approaches, and deepen our understanding of drug resistance mechanisms, ultimately improving survival rates and quality of life for cancer patients. Although most tumor patients initially exhibit a high rate of partial or complete response to standard chemotherapy regimens, the prevalence of drug resistance and poor prognosis tends to increase as chemotherapy continues. Therefore, new approaches are needed to determine effective treatment options. Organoid technology holds significant promise for personalized therapy. By using patient-derived organoid models, the efficacy of different drugs can be evaluated for specific patients, aiding in the development of personalized treatment plans. For instance, a 59-year-old patient with recurrent high-grade serous ovarian carcinoma (HGSOC), who underwent staging surgery following neoadjuvant therapy, exhibited platinum resistance after treatment with paclitaxel, cisplatin, and bevacizumab. Ascites from the patient were used to establish organoids, and subsequent drug sensitivity testing revealed that the chemotherapy drug topotecan and the targeted therapy drug niraparib had stronger inhibitory effects on tumor cells. Based on these results, a final treatment plan of carboplatin, topotecan, and bevacizumab was formulated. After three treatment cycles, CA125 levels decreased, peritoneal effusion was significantly reduced, and the pelvic metastatic tumor showed partial reduction, demonstrating the pivotal role of organoids in precision medicine [ 61 ]. Similarly, Gray et al. [ 62 ] identified the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib and the EGFR inhibitors afatinib and erlotinib as potential treatments for a platinum-resistant stage IIIC low-grade serous ovarian carcinoma (LGSOC) patient who had failed standard chemotherapy and undergone two surgeries. Drug sensitivity analysis using CLIA-certified organoid cultures derived from the patient’s tumor indicated significant clinical improvement after the patient received daily ibrutinib monotherapy for 65 weeks. However, as CA125 levels continued to rise, the treatment was switched to afatinib for 24 weeks, followed by erlotinib due to anemia and the continued increase in CA125 levels. The patient’s intestinal obstruction resolved, pain medication was discontinued, and the ECOG score improved from 3 to 0. This case underscores the clinical utility of in vitro drug testing with patient-derived tumor organoids as a novel precision medicine approach to identify effective personalized treatments for patients who have not responded to standard therapies.

Tissue-derived organoids, are cultivated by embedding stem or progenitor cells within an extracellular matrix (ECM) under conditions that mimic the stem cell niche signaling environment. This review primarily focuses on the construction of organoids from tissue-derived stem cells [ 33 – 43 ]. cells are isolated from tissues obtained from clinical patients. The acquisition of all tissue samples requires written informed consent from patients and approval from an ethics committee. After the patient’s living tissue is excised, it is thoroughly washed to remove blood stains from the surface. The tissue is then cut into approximately 0.5 mm³ pieces and digested using tissue-specific digestive enzymes. The pellet is suspended in the ECM, typically a commercially available matrix such as Matrigel, Cultrex, Geltrex, or BME [ 44 ]. The mixture is incubated at 37 °C, with 5% CO2 and 95% humidity, allowing the matrix to solidify before the culture medium is added. The efficiency of organoid formation can vary, and during passage, organoids are broken down into 3–5 cell clusters, which are then re-suspended in the ECM for further cultivation (Fig.  3 ). Fig. 3 Steps for establishing organoid models of normal and diseased human reproductive organs Steps for establishing organoid models of normal and diseased human reproductive organs The key to successful organoid formation lies in the selection of an appropriate culture medium, without which stem or progenitor cells may struggle to aggregate into clusters, leading to cell death due to the loss of intercellular signaling. Because the signaling pathways that maintain stem or progenitor cells in the reproductive system are not fully understood, the culture medium is typically based on the basic components required for organoid development in human tissues. These components include the tyrosine kinase receptor activator EGF, the WNT signaling pathway activator R-spondin-1, and the BMP signaling pathway inhibitor Noggin. TGF-β signaling pathway inhibitors such as A83-01 may also be included, along with various cytokines, small molecule inhibitors, and amino acids, to form a reproductive system organoid culture medium [ 45 , 46 ]. Currently, the culture medium for ovarian cancer organoids is the most complex. Some researchers have proposed that high-grade serous ovarian cancer (HGSOC) requires low Wnt pathway activity and active BMP signaling [ 36 ], while others suggest selecting a culture medium based on whether the organoids have an active Wnt pathway [ 33 ]. Additional studies have identified NRG1 as a key factor in organoid formation [ 34 ], but challenges remain in optimizing organoid culture media (Tables  1 and 2 ). Table 1 An overview on establishment of human organoids from healthy and diseased human with different culture types in recent years FRT Disease Highlights Organoid Medium Reference Overy Overy • Long-term expandable organoids from human ovarian surface epithelium and ovarian cancer • A platform for ovarian research to describe characterization, biobanking, xenografting, drug screening WNT3A, Noggin, Rspo1, B27, NAC, Primocin, NICO, A83-01, Heregulinβ−1, Y27632, EGF, Forskolin, Hydrocortistone, β-Estradiol Kopper et al., 2019 [ 33 ] Ovarian Cancer WNT3A*, Noggin, Rspo1, B27, NAC, Primocin, NICO, A83-01, Heregulinβ−1, Y27632, EGF, Forskolin, Hydrocortistone, β-Estradiol, FGF-10 Ovarian Cancer - Long-term expandable organoids from human ovarian cancer - NRG1 is a key factor in maximization of OC organoids - A platform for ovarian research to describe characterization, biobanking, drug screening L-glutanine, Penicillin–Streptomycin, A83-01, NICO, N2, B27, NAC, p38i, EGF, Noggin, Rspo1, IGF-1, HGF, NRG1, Y27632, β-Estradiol Maenhoudt et al., 2020 [ 34 ] Ovarian Cancer • Long-term expandable organoids from human ovarian cancer • A platform for ovarian research to describe characterization, biobanking, drug sensitivity and resistance Glutamax, B27, Leu-15-gastrin-1, NAC, IGF-1, FGF-2, WNT3A, Noggin, Rspo1, Y27632, Penicillin–Streptomycin Nanki et al., 2020 [ 35 ] Ovarian Cancer - Long-term expandable organoids from HGSOC cancer require a low-Wnt and an active BMP signaling environment BMP-2, EGF, Y23632, SB431542, B27, N2, NICO, Glutamax, Penicillin–Streptomycin Hoffman et al., 2020 [ 36 ] Endometrium Healthy Endometrium • Long-term expandable organoids from human endometrium • Organoids reflect the physiological response of endometrial epithelium to hormones Penicilin-Streptomycin, Glutamax, B27, N2, Insulin Transferrin Selenium, NICO, EGF, FGF-10, Noggin, Rspo1, A83-01, NAC, SB202190, β-Estradiol Boretto et al., 2017 [ 37 ] Healthy Endometrium - Long-term expandable organoids from human endometrium and endometrial cancer - Organoids reflect the physiological response of endometrial epithelium to hormones, develop characteristics of early pregnancy N2, B27, Primocin, NAC, L-glutamine, EGF, Noggin, Rspo1, FGF-10, HGF, A83-01, NICO Turco et al., 2017 [ 38 ] Healthy Endometrium Eutopic Endometrium Ectopic Endometrium Hyperplastic Endometrium • Long-term expandable organoids from human healthy, eutopic, ectopic and hyperplastic endometrium and endometrial cancer • A platform for endometrial research to describe characterization, biobanking, drug screening Penicilin-Streptomycin, Glutamax, B27, N2, Insulin Transferrin Selenium, NICO, EGF, FGF-10, Noggin, Rspo1, A83-01, NAC, SB202190, β-Estradiol, bFGF, Y27632 Boretto et al., 2019 [ 39 ] Endometrial Cancer Penicilin-Streptomycin, Glutamax, B27, N2, Chemically defined lipid concentrate, NICO, EGF, Noggin, Rspo1, A83-01, NAC, SB202190, β-Estradiol, Y27632, IGF-1, HGF, Il-6 Fallopian Tube Fallopian Tube - Long-term expandable organoids from human fallopain tube rely on Wnt and Notch paracrine signalling WNT3A, Rspo1, Glutamax, B27, N2, EGF, Noggin, FGF-10, NICO, Y27632, SB431542 Kessler et al., 2015 [ 40 ] Cervix Ectocervix • Long-term expandable organoids from human ecto-and endocervix • Organoids depend on opposite Wnt signals Glutamax, B27, N2, Hydrocortistone, EGF, Niggon, FGF-10, NAC, NICO, Y27632, Forskolin, Penicilin-Streptomycin, TGF-β receptor kinase Inhibitor IV Chumduri et al., 2021 [ 41 ] Endocervix Glutamax, B27, N2, Hydrocortistone, EGF, Niggon, FGF-10, NAC, NICO, Y27632, Penicilin-Streptomycin, TGF-β receptor kinase Inhibitor IV, WNT3A, Rspo1 Ectocervix - Long-term expandable organoids from human ecto-and endocervix and cervical cancer - A platform for cervical research to describe characterization, biobanking, xenografting, drug screening Niggon, NICO, p38i, B27, Y27632, NAC, A83-01, FGF-7, Forskolin, FGF-10, Rspo1 Lõhmussaar et al., 2021 [ 42 ] Endocervix Niggon, NICO, p38i, B27, Y27632, NAC, A83-01, FGF-7, Forskolin, FGF-10, Rspo1, EGF, β-Estradiol, WNT activators(WNT surrogate and Chir99021) Cervical Cancer Niggon, NICO, p38i, B27, Y27632, NAC, A83-01, FGF-7, Forskolin, FGF-10, Rspo1 Cervical Cancer • The first establishment of cervical clear cell carcinoma organoids. • A platform for cervical research to describe characterization, biobanking, xenografting, drug screening EGF, Rspol, Noggin, Y27632, Jagged-1, L-glutamine solution, Penicilin-Streptomycin, Amphotericin B suspension Maru, Y et al., 2019 [ 43 ] Vagina NA * With or without WNT3A to select a culture medium suitable for organoid growth OC ovarian cancer, HGSOC high-grade serous ovarian cancer An overview on establishment of human organoids from healthy and diseased human with different culture types in recent years • Long-term expandable organoids from human ovarian surface epithelium and ovarian cancer • A platform for ovarian research to describe characterization, biobanking, xenografting, drug screening - Long-term expandable organoids from human ovarian cancer - NRG1 is a key factor in maximization of OC organoids - A platform for ovarian research to describe characterization, biobanking, drug screening • Long-term expandable organoids from human ovarian cancer • A platform for ovarian research to describe characterization, biobanking, drug sensitivity and resistance • Long-term expandable organoids from human endometrium • Organoids reflect the physiological response of endometrial epithelium to hormones - Long-term expandable organoids from human endometrium and endometrial cancer - Organoids reflect the physiological response of endometrial epithelium to hormones, develop characteristics of early pregnancy Healthy Endometrium Eutopic Endometrium Ectopic Endometrium Hyperplastic Endometrium • Long-term expandable organoids from human healthy, eutopic, ectopic and hyperplastic endometrium and endometrial cancer • A platform for endometrial research to describe characterization, biobanking, drug screening • Long-term expandable organoids from human ecto-and endocervix • Organoids depend on opposite Wnt signals - Long-term expandable organoids from human ecto-and endocervix and cervical cancer - A platform for cervical research to describe characterization, biobanking, xenografting, drug screening • The first establishment of cervical clear cell carcinoma organoids. • A platform for cervical research to describe characterization, biobanking, xenografting, drug screening * With or without WNT3A to select a culture medium suitable for organoid growth OC ovarian cancer, HGSOC high-grade serous ovarian cancer Table 2 Details of the establishment of different types of organoids, including tissue digestive enzymes, extracellular matrix, passage ratio, morphology, and identification methods Organoids Tissue sourse Enzymoilsis ECM Passage/Ratio Phenotype Main ways of identification OSE-O Ovarian tissue(undergoing prophylactic bilateral salpingo-oophorectomy) Collagenase(0.5–1 mg/ml, Clostridium histolyticum) BME 2-3weeks 1∶1–4 Cystic Marker OC-O Epithelial ovarian carcinoma(LGS, MC, END, CCC, HGS) Collagenase(0.5–1 mg/ml, Clostridium histolyticum) or collagenase I and collagenase II(1∶1) or Collagenase IV or collagenase I, dispase II, DNase I BME or Matrigel 1-4weeks 1∶1–4 Denser organoid structures harboring multiple lumens(LGS, MC, END, CCC); cystic to dense with different degrees of circularity and cellular cohesiveness(HGS) Marker; Gene expression profiles EM-O Healthy endrometeium(a hysterectomy performed for benign uterine diseases) 1.25 U/ml Dispase II/0.4 mg/ml collagenase V or collagenase IV(1 mg/ml) Matrigel 14days 1∶3–5 Cystic Marker; Hormone response EUT/ECT-O Ectopic (stages I–IV) and eutopic endometrium(endometriosis Biopsies) Collagenase IV(1–2 mg/ml) Matrigel 7-10days NA Cystic(thicker, luminal invasion, stratified epithelium) Marker; Gene expression profiles EC-O Endometrial cancer(different grades and progression stages) Collagenase IV(1–2 mg/ml) Matrigel 7-20days NA Glandular-like morphology with a well-to-moderately defined lumen(low-grade/stage cancer); dense without a visible lumen(high-grade/stage cancer) Marker; Gene expression profiles FT-O Anatomically normal fallopian tubes(standard surgical procedures for benign gynecological disease) Collagenase I(0.5 mg/ml) Matrigel 2-3weeks 1∶3 Cystic Marks Cervical-O healthy cervical tissue(a hysterectomy performed for benign uterine diseases) Collagenase II(0.5 mg/ml) or Collagenase(1 mg/mL, Clostridium histolyticum) Matrigel or BME 2-3weeks 1∶10(ectocervix) 1∶5(endocervix) Polarized monolayered architecture(endocervix); stratified multilayered phenotype(ectocervix) Marker; Gene expression profiles Cervical tumoroids Cancer tissue (SqCa, AdCa and CCC) Collagenase(1 mg/mL, Clostridium histolyticum) or 2 U/ml Disase II、1 mg/mL collagenase P and Accumax Matrigel or BME NA Loss of stratification and poor cellular polarity(SqCa); denser structures with prominent vacuolization(AdCa) Marker; Gene expression profiles ECM extracellular matrix, BME basement membrane extract, SqCa squamous cell carcinoma, AdCa adenocarcinoma, LGS low-grade serous, MC mucinous, END endometrioid, CCC clear cell, HGS high-grade serous, OSE-O ovarian surface epithelium organoid, EM endrometeium, EUT/ECT eutopic/ectopic, EC endometrial cancer, FT fallopian tube Details of the establishment of different types of organoids, including tissue digestive enzymes, extracellular matrix, passage ratio, morphology, and identification methods 1-4weeks 1∶1–4 14days 1∶3–5 7-10days NA 7-20days NA 2-3weeks 1∶3 2-3weeks 1∶10(ectocervix) 1∶5(endocervix) ECM extracellular matrix, BME basement membrane extract, SqCa squamous cell carcinoma, AdCa adenocarcinoma, LGS low-grade serous, MC mucinous, END endometrioid, CCC clear cell, HGS high-grade serous, OSE-O ovarian surface epithelium organoid, EM endrometeium, EUT/ECT eutopic/ectopic, EC endometrial cancer, FT fallopian tube The identification of gynecological disease organoids can be achieved through various methods, including histological, functional, and genetic testing, to ensure the reliability and efficacy of organoids in research and clinical applications. Organoids derived from normal reproductive system tissues typically exhibit a single-layer cystic structure. Organoids derived from extracevical tissues display layered or multilayered phenotypes, whereas malignant tumor organoids can present a range of phenotypes, from cystic to dense, often with varying degrees of nuclear abnormalities, unclear structures, loss of stratification, poor cell polarity, and absence of lumen formatio [ 33 – 42 ]. Immunofluorescence staining is employed to detect the expression and localization of specific proteins, allowing for the identification of cell types and their statuses within organoids (Fig.  4 ). Fig. 4 Organoid models derived from normal and diseased tissues of the human FRT Organoid models derived from normal and diseased tissues of the human FRT Normal endometrial organoids retain their hormonal responsiveness, exhibiting menstrual cycle-like changes in vitro when stimulated by estrogen and progesterone. During the secretory phase, increased expression of glycogen, PAEP, and SPP1 prepares the endometrium for embryo implantation. Upon exposure to pregnancy signals, endometrial organoids acquire characteristics typical of early pregnancy [ 38 ]. Cervical organoids can respond to human papillomavirus (HPV) infection, simulating the progression of HPV-related cervical cancer [ 42 ]. The sensitivity of malignant tumor organoids to chemotherapy drugs has been shown to be similar or even superior to that of cell line models and animal models in terms of biological behavior and drug response [ 33 , 39 , 42 ]. Gene detection in organoids is often conducted using RNA sequencing (RNA-Seq) technology to analyze gene expression profiles. This method not only assesses the similarity of organoid gene expression to the original tissue but also reveals changes in gene expression in organoids under different conditions. For instance, endometrial cancer-related genes such as PTEN and PIK3CA [ 39 ], cervical cancer-related genes like TP63 and MKI67 [ 42 ]., and ovarian cancer-related genes including PAX8 and p53 [ 33 ] have been studied. Additionally, single-cell RNA sequencing can analyze gene expression at the single-cell level within organoids, providing high-resolution data on cell heterogeneity and uncovering the gene expression characteristics of different cell types and their interrelationships within organoids [ 47 ]. These identification methods not only enhance the quality of organoid research but also promote the broad application of organoid technology in the study of gynecological diseases.

Organoids can be derived from either pluripotent stem cells (PSCs) or adult stem cells (ASCs), which can be fetal or adult in origin [ 23 , 24 ]. PSCs-derived organoids are formed by harnessing their inherent ability to differentiate and spatially organize in response to developmental cues, thereby adopting specific cell fates. ASCs-sourced from adult tissues, typically have limited differentiation potential, however, they still play a crucial role in forming functional organoids and facilitating tissue regeneration in vitro (Fig.  2 ). Fig. 2 Organoids can be grown from adult stem cells (ASCs) or pluripotent stem cells (PSCs) Organoids derived from pluripotent stem cells (PSCs) initially require targeted differentiation into specific germ layers (endoderm, mesoderm, or ectoderm). Subsequently, the culture is supplemented with specific growth and signaling factors to induce the formation of desired cell types and organs. In contrast, organoid cultures derived from adult stem cells (ASCs) involve isolating tissue-specific stem cell populations and embedding them in an extracellular matrix (ECM) along with specific growth and signaling factors Organoids can be grown from adult stem cells (ASCs) or pluripotent stem cells (PSCs) Organoids derived from pluripotent stem cells (PSCs) initially require targeted differentiation into specific germ layers (endoderm, mesoderm, or ectoderm). Subsequently, the culture is supplemented with specific growth and signaling factors to induce the formation of desired cell types and organs. In contrast, organoid cultures derived from adult stem cells (ASCs) involve isolating tissue-specific stem cell populations and embedding them in an extracellular matrix (ECM) along with specific growth and signaling factors Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have the capacity to differentiate into multiple cell types from all three germ layers (ectoderm, mesoderm, and endoderm) under controlled conditions, making them a valuable tool for organoid construction [ 25 ]. By modulating growth factors and signaling pathways, ESCs and iPSCs can be directed to form organoids representing specific organs. Yucer et al. [ 26 ], demonstrated the directional differentiation of iPSCs into intermediate mesoderm (IM) of tubal origin by adding CHIR99021, activin A, and BMP4. Subsequently, activation of WNT4 followed by Follistatin promotes IM differentiation into Müllerian ducts and the female reproductive tract (FRT) rather than nephron structures. Due to the reprogramming process, iPSCs may retain cellular stress or genetic variations, which could impact their function and safety. Therefore, rigorous quality control of iPSCs is essential to ensure genomic stability and phenotypic consistency. In contrast, ASCs are sourced directly from specific tissues and grown under conditions that mimic native stem cell niche signals. ASCs retain microenvironmental characteristics similar to their tissues of origin, which may enhance their potential to form functional organoids. For example, intestinal organoids derived from stem cells in intestine preserve the self-renewal and differentiation capabilities of intestinal stem cells and can model intestinal absorption, secretion, and other functions in vitro [ 27 ]. Similarly, lung organoids derived from adult lung stem cells facilitate the study of respiratory diseases, including chronic obstructive pulmonary disease (COPD), asthma, lung cancer, and pulmonary fibrosis [ 28 , 29 ]. Additionally, generating organoid models from stem cells obtained from patients with genetic diseases enables the study of tissue-specific functional defects, advancing understanding of the molecular mechanisms of these disorders and aiding in the search for potential treatments. The regenerative potential of stem cells offers significant promise for the application of stem cell-derived organoids in transplantation. Organoids generated from a patient’s own stem cells enable personalized medicine, minimize immune responses, and improve transplant success rates. For example, intestinal organoids can repair damage caused by inflammation or trauma, with potential applications for treating diseases [ 30 ]. Liver organoids, derived from liver stem cells, represent a promising alternative to liver transplantation and may support the regeneration of damaged liver tissue [ 31 ]. Similarly, skin organoids generated from skin stem cells hold important potential for treating burns and other dermatological injuries [ 32 ]. Additionally, organoids developed from ovarian and endometrial stem cells are emerging as a focal area in transplantation and regenerative medicine, offering new possibilities for treating reproductive system disorders, including conditions such as endometrial injury and ovarian function decline. Despite technical and ethical challenges, the future of stem cell-derived organoids in transplantation is promising.

Tumor tissue xenotransplantation is a widely used research method in which tumor tissues are transplanted into immunodeficient mice to observe and manage them in a controlled environment, including conducting drug therapy experiments and monitoring tumor growth [ 63 ]. Bonazzi et al. [ 64 ] directly transplanted endometrial cancer tissue into mice. The resulting patient-derived xenograft (PDX) model exhibited four molecular subtypes and successfully captured the intratumoral heterogeneity of the original primary tumor. PDXs of the copy-number high subtype were sensitive to PARP inhibitors, suggesting that this heterogeneity could be leveraged to optimize treatment and improve patient prognosis. However, maintaining mouse models and conducting long-term experiments can be costly and time-consuming, particularly when large numbers of mice are required or when experiments are complex.Kopper et al. [ 33 ] developed OC organoids that not only verified drug sensitivity but also could be transplanted in situ or subcutaneously into immunodeficient mice, allowing for in vivo drug sensitivity analysis. In 2021, Berg et al. [ 59 ] demonstrated that EC organoids and organoid derived PDX (O-PDX) models replicated the tissue structure, protein biomarker expression, and genetic profile of the original tissues. Even O-PDX-derived organoids exhibited stable mutational profiles. The heterogeneity and drug sensitivity of organoids were comparable to those of the O-PDX model. These results support the use of organoids for initial in vitro drug screening, followed by in vivo validation and systemic drug effect testing in the O-PDX model. In 2023, Liu et al. [ 65 ] obtained tumor samples from cervical cancer patients and transplanted them into immunodeficient mice, successfully establishing a highly faithful PDX model that accurately reflected the morphology, gene expression, and molecular characteristics of the original tumors. A PDX-derived organoid (PDXO) model was created using early PDX tumor tissues, both of which retained the HER2 mutation sites and the histological features of the corresponding original tumor. Both models demonstrated that the combination of HER2 inhibitors and tumor-infiltrating lymphocytes (TILs) could be an effective strategy for treating cervical cancer patients with HER2 mutations. The combination of organoids and mouse models offers a novel preclinical approach for disease exploration and treatment (Fig.  5 ). Fig. 5 The combination of organoids and mouse models provides a novel preclinical model for disease drug screening. PDX patient-derived xenograft, O-PDX organoid derived PDX, PDX-O PDX-derived organoid, PDOs patient-derived organoids The combination of organoids and mouse models provides a novel preclinical model for disease drug screening. PDX patient-derived xenograft, O-PDX organoid derived PDX, PDX-O PDX-derived organoid, PDOs patient-derived organoids

The cultivation and maintenance of organoids continue to face significant technical challenges, particularly concerning complex reproductive organs. The development of organoids is highly dependent on the cell type and the state of the original tissue, leading to potential variability in organoid morphology and function due to individual differences. Prolonged culture periods can result in issues such as cellular aging, genetic mutations, and abnormal differentiation, thereby posing challenges to experimental reproducibility and standardization. Currently, most organoids tend to overgrow, lacking essential matrix components, immune cells, and functional vasculature [ 95 ]. The excessive proliferation of epithelial cells disrupts the balanced growth of other cell types, fails to accurately simulate the diversity and complexity of actual tissues, obscures critical physiological and pathological characteristics of other cell types, and potentially biases research outcomes, thereby diminishing their application value in disease research. Additionally, organoid models devoid of immune cells cannot fully replicate the immune environment and pathological processes in the human body, which is crucial for the study of infectious diseases and cancer immunotherapy [ 96 ]. Finally, the absence of functional vasculature in organoids not only limits their growth and metabolic functions but also restricts their utility as platforms for drug screening and toxicology research. Future research must focus on improving culture conditions, enhancing multi-cell type co-culture, and developing technologies that support the formation of functional vasculature to enhance the physiological and pathological relevance of organoids, thereby advancing their application in medical research and clinical application.

Stem cells have shown significant potential in organoid applications, leading to unprecedented breakthroughs and opportunities in medical research and treatment, offering new possibilities for personalized therapy, drug screening, disease modeling, and regenerative medicine. However, its widespread application still encounters challenges, including high technical costs, specific laboratory requirements, technical barriers, and inherent model limitations. As technology continues to advance, these challenges are expected to be overcome, thereby further enhancing the application and development of organoids in the medical field and bringing increased innovation and hope to the research and treatment of gynecological diseases.

Organ-on-a-chip is a miniaturized platform that utilizes microfluidic technology to culture cells on a microchip, simulating the microenvironment and functional characteristics of human organs, including cell-cell interactions, tissue structure, and physiological functions [ 66 – 69 ]. In 2021, Ahn et al. [ 70 ] developed the first microengineered vascularized endometrial organ chip containing three types of cells: epithelium, mesenchyme, and blood vessels. This model successfully summarized endometrial angiogenesis and hormone response in vivo, revealing key features of the proliferative and secretory phases of the menstrual cycle. The model also demonstrated that the emergency contraceptive levonorgestrel had dose-dependent effects on endometrial transmission and vascular regression. Microfluidic-based organ-on-a-chip technology provides a physiologically relevant, organ-specific test platform capable of simulating the in vivo microenvironment. Izadifar et al. [ 71 ] established a microfluidic organ-on-a-chip model of human cervical mucosa with a epithelial-mesenchymal interface, which produced abundant mucus and exhibited biophysical and hormonal responses. Cervical microchips were co-cultured with Lactobacillus and Gardnerella vaginalis communities to simulate the effects of optimal (healthy) or non-optimal (pathogenic) microbiome interactions associated with female reproductive tract infections. Similarly, Mahajan et al. [ 72 ] explored human vagina-microbiome interactions using a vaginal organ-on-a-chip model. Park et al. [ 73 ] developed a dual reproductive organ chip with bidirectional endocrine crosstalk between the endometrium and ovary, identifying SERPINB2 as a marker of reproductive toxicity that significantly increased in response to various toxic exposures. This organ chip not only reflects the multicellular complexity of the human female reproductive tract but also provides greater accuracy in evaluating the reproductive toxicity of drug candidates. Organoids and organ-on-a-chip are two distinct yet complementary methods for replicating human organs in vitro. Organoids-on-a-chip combines the strengths of both approaches to create a more powerful in vitro technology, addressing issues such as incompatible media and multi-level, multi-component culture, and providing a highly precise model for research [ 74 , 75 ]. Yin et al. [ 76 ] used organoids-on-a-chip technology to develop a hepato-cardiac organoid model derived from hiPSCs, enabling hepato-cardiac co-culture for drug metabolism and toxicity testing. Tao et al. [ 77 ] constructed a hepato-islet organoid model derived from hiPSCs using organoids-on-a-chip technology to simulate liver-islet interactions in carbohydrate metabolism. Combining primary or stem cell-derived human reproductive organoids with other organoids on a chip will yield more reliable data for developing more effective and safer drugs and therapeutic strategies for precision medicine. Research on organoid chips for gynecological diseases has not yet been reported. However, with the continuous advancement of technology, the application of organoid chips in gynecological diseases is expected to expand, potentially revolutionizing research and treatment in this field. 3D printing technology enables the rapid and precise fabrication of complex tissue structures, including blood vessels and microstructures, thereby facilitating more accurate simulations of human tissues [ 78 , 79 ]. Wu et al. [ 80 ] utilized GelMA bio-ink to develop 3D-printed ovaries capable of supporting the growth and maturation of mouse follicles. In a related study, Laronda et al. [ 81 ] implanted artificial ovaries composed of 3D-printed scaffolds and follicles into surgically sterilized mice. These scaffolds became vascularized, leading to the full restoration of ovarian function and successful delivery of healthy pups. Zhao et al. [ 82 ] applied 3D printing technology to construct an in vitro cervical tumor model using Hela cells embedded in a gelatin/alginate/fibrinogen hydrogel. Within this 3D-printed environment, Hela cells formed spheroids and maintained a high proliferation rate, better simulating tumor characteristics compared to 2D-cultured cells. Traditional organoid culture models fail to replicate mature organ structures and their associated tissue microenvironments. In contrast, 3D printing technology allows for the design of specific structures that facilitate close cellular interactions [ 83 – 85 ]. Brassard et al. [ 86 ] advanced this field by using organoids deposited directly into the extracellular matrix to generate centimeter-scale tissues with crypt and villus domains, achieved by controlling geometry and cell density. Similarly, Kim et al. [ 87 ] engineered a multi-layered bladder “assembly” that successfully replicated the structural and molecular functions of bladder tissue through the recombination of tissue stem cells and matrix components. However, research on 3D printing for gynecological disease organoids remains limited. Challenges such as the compatibility of 3D-printed biomaterials with cells, damage caused by extrusion pressure during printing, and cell density continue to impede the advancement of in vitro reproductive organ printing.

The female reproductive system comprises the ovaries, fallopian tubes, endometrium, cervix, and vagina, with its proper function being essential for female fertility and overall health [ 1 ]. Gynecological diseases encompass a variety of conditions affecting the female reproductive system, including common ones such as vaginitis, cervicitis, endometriosis, ovarian cysts, uterine fibroids, and cancers of the endometrium, cervix, and ovaries [ 2 , 3 ]. The complex pathological mechanisms and diverse clinical manifestations of these diseases present significant challenges to their treatment. To enhance the understanding of the occurrence, development, and treatment of these diseases, scientists have developed various research models, including in vitro cell models and animal model [ 4 , 5 ]. In vitro cell models are particularly important in studying gynecological diseases. By cultivating primary cells or cell lines, researchers can replicate the microenvironment of these diseases and observe processes such as cell proliferation, migration, and differentiation. For example, Li et al. found that the overexpression of FPN could reduce Erastin-induced ferroptosis in endometrial stromal cells, suggesting a novel approach for treating endometrial disorders [ 6 ]. Although in vitro models provide valuable insights, they cannot fully replicate the complex physiological environment found in vivo. Animal models also play a crucial role in gynecological disease research. Species such as mice, rats, rabbits, and non-human primates are commonly used to model the pathological processes of human gynecological diseases. Greaves et al. [ 7 ] developed a mouse model of endometriosis that closely resembles human peritoneal lesions in terms of estrogen receptor expression, inflammation, and macrophage infiltration. Similarly, Topp et al. [ 8 ] created human HGSOC patient-derived xenografts (PDXs) by transplanting fresh HGSOC tissue into subcutaneous and ovarian sites in mice, achieving the same “platinum response” observed in patients. However, the species differences inherent in animal models limit the direct applicability of findings to humans. Stem cells possess the ability to self-renew and differentiate into various functional cell types, however, traditional in vitro stem cell cultures, which are based on 2D systems, often fail to stably maintain stem cell properties and fully induce their differentiation potential. In 2009, Hans Clevers et al. successfully cultured Lgr5 + intestinal stem cells into three-dimensional structures with crypt-like and villous epithelial regions in vitro, marking the advent of organoid research [ 9 ]. Subsequently, human liver and kidney organoids were also successfully established [ 10 , 11 ]. The application and development of organoids in disease research have demonstrated great potential. Organoids can replicate the structure and function of organs in vivo, more accurately simulate disease microenvironments, and more faithfully reflect disease states, providing powerful tools for studying cell behavior, signaling pathways, and drug screening [ 12 – 17 ]. For instance, Vlachogiannis et al. [ 18 ] demonstrated that organoids have 100% sensitivity, 93% specificity, an 88% positive predictive value, and a 100% negative predictive value, making them highly effective in predicting patient responses to anticancer drugs. Organoids also hold promise in tissue engineering and regenerative medicine. Yui et al. [ 19 ] successfully regenerated functional and histologically normal self-renewing crypts by cultivating GFP + colonic organoids and retransplanting them into the colons of mice with superficial injuries. Furthermore, genetic engineering and the use of functional biomaterials can enhance the success rate of organoid transplantation, offering new avenues for the treatment and repair of human organ lesions [ 12 ]. Currently, the development of organoids for gynecological diseases is well underway, with research being conducted in various fields [ 20 – 22 ]. In this review, we provide a comprehensive overview of recent advances in organoids derived from adult female reproductive tissues, focusing on their application in drug screening, disease modeling, transplantation, and regenerative medicine (Fig.  1 ). Fig. 1 Various biomedica applications of organoid model systems Various biomedica applications of organoid model systems

Infertility and hormone deficiency are significant consequences of organ and tissue dysfunction in the female reproductive system, for which traditional treatments often fail to fully cure or repair the damaged tissues and organs. Regenerative medicine aims to restore the function of these tissues and organs through the repair, regeneration, or replacement of damaged parts using retransplantation of fresh or cryopreserved organs and tissue engineering techniques, including cells, growth factors, and biomaterials [ 88 – 91 ]. In 2004, Donnez et al. [ 92 ] reported the case of a girl with Hodgkin lymphoma who underwent ovarian tissue cryopreservation before chemotherapy and later received orthotopic autologous transplantation of the preserved ovarian tissue, successfully giving birth to a baby girl. To date, there have been at least 130 similar reports [ 93 ]. Laronda et al. [ 81 ] implanted artificial ovaries into surgically sterilized mice, resulting in the complete restoration of ovarian function and the birth of offspring following natural mating. However, the clinical application of artificial ovaries remains limited due to ethical, technological, and biomaterial challenges. Organoids, as bioengineering tools, offer potential for studying physiological mechanisms, diagnostic purposes, and therapeutic methods, thus providing hope for the development of artificial reproductive organs. Zhang et al. [ 94 ] transplanted mouse endometrial organoids into an intrauterine adhesion (IUA) model in mice, promoting endometrial repair and regeneration, which subsequently allowed successful mating and the birth of healthy offspring. The advancement of this technology could serve as a valuable model for human endometrial repair. Combining organoids with microfluidic and 3D printing technologies presents promising prospects for the development of artificial reproductive organs.