3D bioprinting technology innovation in female reproductive system.

Due to ethical concerns surrounding the use of human subjects in biomedical research [ 123 ] most basic research utilizes in vivo animal models and 2D cell/tissue culture models to study the female reproductive system [ 124 ]. However, the complexity and species-specific differences in the female reproductive system make direct comparisons with animal models challenging. Additionally, reproductive organs in the human body do not function in isolation; they are interconnected with other organs to maintain reproductive and endocrine functions. Single-layer cells cultured on flat plastic or glass lose their three-dimensional structure and physical or biochemical interactions with other cells in the body [ 125 ]. These limitations highlight the urgent need for structural models that accurately replicate the physiological microenvironment of the human female reproductive system. The three-dimensional distribution of cells in 3D-bioprinted in vitro models more closely resembles the in vivo situation, better simulating cell–cell and cell–ECM interactions within the female reproductive system. Particularly in research on tumors of the female reproductive system, 3D-bioprinted in vitro models can replicate tumor heterogeneity and its relationship with the microenvironment, providing a foundation for studying tumor pathology and screening potential drugs [ 126 ]. Below is a review of research on 3D bioprinting for constructing models of female reproductive system tumors and other pathological or physiological conditions. Cervical cancer is the third most common malignant tumor among women worldwide [ 125 ] . It can be classified into three pathological types: squamous cell carcinoma, adenocarcinoma, and adenosquamous cell carcinoma [ [127] , [128] ] . Persistent infection with high-risk HPV is the primary risk factor for cervical cancer, with over 90 % of cases associated with high-risk HPV infection [ 129 ] . Surgical treatment is the standard approach for early-stage cervical cancer, and synchronous radiotherapy and chemotherapy are performed for patients with high risk of recurrence pathological factors after surgery; There is no consensus on adjuvant therapy for patients with intermediate risk factors, and Sedlis criteria are commonly used in clinical practice. The standard treatment for locally advanced cervical cancer is concurrent radiotherapy and chemotherapy, with a cure rate of up to 60 %. Despite standard treatment, 30 % of patients still experience local recurrence or metastasis, resulting in a low survival rate and being the main cause of death at present. The treatment options for advanced or recurrent cervical cancer remain limited, with poor prognosis posing a significant challenge in cervical cancer treatment [ 128 ] . In 2014, Zhao et al. [ 4 ] constructed an in vitro cervical tumor spheroid model with tumorigenic properties using a gelatin/fibrinogen/alginate hydrogel loaded with HeLa cells via EBB ( Fig. 9 ). This model was used to study cell proliferation, matrix metalloproteinases (MMPs), and responses to paclitaxel treatment. Compared with traditional 2D culture models, HeLa cells in the 3D model showed enhanced proliferation, migration abilities, and higher paclitaxel resistance. This study pioneered the use of 3D bioprinting to construct a cervical cancer model in vitro and comprehensively compared 2D and 3D cervical cancer models concerning cell proliferation, metastasis, and drug resistance, demonstrating that the 3D model effectively mimics in vivo tumor characteristics. Additionally, the study provides detailed insights into constructing an in vitro cervical cancer model using 3D bioprinting, establishing a theoretical and practical foundation for this technology's application in cervical cancer tumor biology research. Fig. 9 Three-dimensional printing of HeLa cells for cervical tumor model in vitro. A The schematic of 3D HeLa/hydrogel constructs. B. Top view of 3D HeLa/hydrogel constructs on day 0, day 5, and day 8. C. Cellular morphological changes during 8 days of culture. D. MMP secretion of HeLa cells in 3D constructs and 2D planar culture. E. Chemoresistance of HeLa cells in 3D HeLa/hydrogel constructs and 2D planar culture. Reproduced from Ref. [ 4 ], with permission of 2014 IOP Publishing Ltd. Fig. 9 Three-dimensional printing of HeLa cells for cervical tumor model in vitro. A The schematic of 3D HeLa/hydrogel constructs. B. Top view of 3D HeLa/hydrogel constructs on day 0, day 5, and day 8. C. Cellular morphological changes during 8 days of culture. D. MMP secretion of HeLa cells in 3D constructs and 2D planar culture. E. Chemoresistance of HeLa cells in 3D HeLa/hydrogel constructs and 2D planar culture. Reproduced from Ref. [ 4 ], with permission of 2014 IOP Publishing Ltd. In 2018, a study [ 130 ] based on Zhao et al.'s methods developed a model of advanced cervical cancer using a gelatin/fibrinogen/alginate hydrogel loaded with HeLa cells. Upon TGF-β supplementation, HeLa cells aggregated and began to disassemble, with some cells acquiring fibroblast-like spindle morphology, indicating induction of epithelial-mesenchymal transition (EMT). The addition of disulfuron and EMT pathway inhibitor C19 inhibited TGF-β-induced EMT in a dose-dependent manner. This study demonstrated TGF-β′s role in inducing EMT within a 3D-bioprinted cervical cancer model, offering a theoretical basis for research on cervical cancer metastasis and treatment. In 2023, another study [ 10 ] used 3D bioprinting to investigate spatial gradients of HeLa cell concentration in high-resolution cervical tumor models. SECM was employed to quantitatively measure drug molecule diffusion over time in the 3D cervical cancer tumor structure. EBB with alginate as the bioink provided the basis for the model, with alginate's printability being influenced by its molecular weight and crosslinking ratio. The study detailed the preparation of biocompatible and printable alginate solutions and optimized conditions to ensure bioink consistency and model fidelity. Using SECM, the study spatially resolved oxygen concentrations within HeLa cell spheroids, leveraging nanoelectrodes and phase-contrast microscopy. Since oxygen levels critically regulate cellular processes and significantly influence cellular behavior under both physiological and pathological conditions, this technique provides valuable insights into how oxygen concentration affects cervical cancer cells. Additionally, SECM has the potential to investigate the spatial distribution of cellular metabolites within spheroids. By evaluating drug penetration and distribution within 3D-bioprinted models, SECM can simulate anti-cancer compound diffusion in tumor clusters, aiding in drug efficacy studies for cervical cancer. Ovarian cancer has the highest mortality rate among gynecological tumors , posing significant threats to women's health and life [ 131 ]. Most cases (70 %) are diagnosed at late stages because the clinical manifestations of early ovarian cancer are relatively insidious and non-specific In recent years, although significant progress has been made in the treatment and research of ovarian cancer, the lack of early diagnosis and the occurrence of postoperative chemotherapy resistance have limited improvements in the 5-year survival rate for patients with ovarian cancer [ 132 ] . The demand for individualized models in ovarian cancer treatment and the development of precision therapies have promoted the creation of 3D-bioprinted tissue models. Xu et al. employed an inkjet-based 3D bioprinting method to print human ovarian cancer cells (OVCAR-5) and MRF-5 cells (a normal human fibroblast cell line) in a controlled spatial distribution atop a Matrigel matrix scaffold, creating a 3D culture model to study the regulatory feedback mechanisms between tumors and matrix cells, as well as for drug sensitivity testing [ 6 ]. Baka et al. [ 133 ] used a gelatin-alginate saline gel loaded with ovarian cancer cells (SKOV-3) and cancer-associated fibroblasts (CAFs) for 3D bioprinting to develop an in vitro model of ovarian tumors. In this model, it was observed that cells self-assemble into heterotypic aggregates, which can be utilized to construct ovarian cancer organoids. Mekhileri et al. [ 134 ] created a macroscale ovarian model by assembling a 3D-printed hydrogel scaffold into a heterogeneous spheroid containing ovarian adenocarcinoma cells and fibroblasts ( Fig. 10 ). Compared with a single spheroid module, the increase in size and complexity of tumors leads to a decrease in sensitivity to chemotherapy drugs. These results indicate that the planar culture model cannot accurately simulate the impact of the physiological microenvironment on drug pharmacokinetics after losing the three-dimensional structure of normal in vivo tissues. Therefore, when comparing 3D-bioprinted organoids with 2D cultures, organoid models exhibit resistance to chemotherapy drugs. Fig. 10 Construction of 3D in vitro model of ovarian cancer. A. Cancer 3D in vitro model overview. B. Live and dead staining cells of reproducible ovarian carcinoma spheroids on day 7 using the liquid overlay method. C. Live and dead staining cells of reproducible ovarian carcinoma GelMA microspheres using a visible-light microfluidic approach. D. Ovarian carcinoma coculture construct bioassembly into PEGT:PBT scaffolds. E. Darkfield images of assembled coculture construct imaged after 4 days of exposure to doxorubicin. Reproduced with permission from Ref. [ 134 ] under Creative Commons license. Fig. 10 Construction of 3D in vitro model of ovarian cancer. A. Cancer 3D in vitro model overview. B. Live and dead staining cells of reproducible ovarian carcinoma spheroids on day 7 using the liquid overlay method. C. Live and dead staining cells of reproducible ovarian carcinoma GelMA microspheres using a visible-light microfluidic approach. D. Ovarian carcinoma coculture construct bioassembly into PEGT:PBT scaffolds. E. Darkfield images of assembled coculture construct imaged after 4 days of exposure to doxorubicin. Reproduced with permission from Ref. [ 134 ] under Creative Commons license. The application of 3D bioprinting in drug screening enhances physiological relevance by creating three-dimensional tissue models that closely mimic the in vivo environment. Therefore,3D-bioprinted cervical and ovarian cancer tumor models have been successfully used to study tumor occurrence and cell response to clinically relevant chemotherapy drugs. This novel in vitro bioprinted tumor model exhibits 3D biological features, making it an important tool for studying 3D tumor biology.This advancement is poised to improve the efficiency and accuracy of high-throughput drug screening. Additionally, it enables personalized drug screening, providing strong support for personalized medicine. Endometriosis is a common disease that affects 178–200 million women worldwide. It refers to the growth of endometrial-like tissue outside the uterine cavity, often invading structures within the pelvic cavity, including the peritoneum, ovaries, bladder, small intestine, and colon. The main clinical manifestations of endometriosis are chronic pelvic pain and infertility [ 135 , 136 ].The lack of representative in vitro models of endometriosis hinders research in this field. In a 2020 study [ 137 ], 3D bioprinting was used to create a three-dimensional biological model of frameless endometriosis using the 12Z endometrial cell line and a phosphate matrix, employing the Kenzan method ( Fig. 11 ). This model expresses high levels of estrogen-related genes and secretes a significant amount of inflammatory factors associated with endometriosis, independent of TNFα stimulation. Additionally, the study constructed a 3D construct with 12Z cells in the periphery and HeyA8 cells in the core, which can be used to investigate the pathogenesis of endometriosis-related ovarian cancer. Furthermore, this study attempts to construct a biosphere using endometrial stromal cells (T-HESCs) and 12Z cells to explore the pathogenesis of endometriosis. Although the 12Z cell line used originates from peritoneal lesions and cannot fully represent the various molecular forms of endometriosis, this biological model is still expected to serve as a conceptual validation basis for studying endometriosis and its microenvironment. Fig. 11 Three-dimensional biofabrication models of endometriosis. A. Kenzan method of biofabrication. a-b. A Kenzan used for 3D biofabrication with the Regenova Bio 3D Printer. c. Workflow of Kenzan method biofabrication. B. Large 12Z spheroids. a-b. The change of large 12Z spheroids at 24 h and 48 h. c-d. 12Z spheroids remain alive for at least 120 h. C. Representative image of a spheroid, which fits all of the Regenova goal parameters. D. 3D biofabrication of 12Z and HEYA8 spheroids into constructs. E. Spheroids made from 12Z and T-HESCs and KRT-7 as a epithelial marker. Reproduced from Ref. [ 137 ] under Creative Commons license (CC-BY). Fig. 11 Three-dimensional biofabrication models of endometriosis. A. Kenzan method of biofabrication. a-b. A Kenzan used for 3D biofabrication with the Regenova Bio 3D Printer. c. Workflow of Kenzan method biofabrication. B. Large 12Z spheroids. a-b. The change of large 12Z spheroids at 24 h and 48 h. c-d. 12Z spheroids remain alive for at least 120 h. C. Representative image of a spheroid, which fits all of the Regenova goal parameters. D. 3D biofabrication of 12Z and HEYA8 spheroids into constructs. E. Spheroids made from 12Z and T-HESCs and KRT-7 as a epithelial marker. Reproduced from Ref. [ 137 ] under Creative Commons license (CC-BY). The uterine muscle layer is the main tissue structure responsible for uterine contractions, which are crucial for various reproductive functions, such as the menstrual cycle, transportation of sperm and embryos, pregnancy, and childbirth [ 138 , 139 ]. Dysregulation of uterine contractility can lead to common pathological diseases, including premature birth, infertility, implantation abnormalities, and irregular menstrual cycles [ [140] , [141] , [142] ]. Uterine contractility is a three-dimensional coordinated phenomenon that should be studied in a three-dimensional environment. Souza et al. [ 143 ] used 3D bioprinting for the first time to print uterine muscle cells from different patient sources into 3D-bioprinted hollow rings, which can be used to study the physiological mechanisms of uterine contractility and the effects of various clinically relevant drugs, such as nifedipine and indomethacin, on uterine contractions.

Siyao Chen: Writing – review & editing, Software, Methodology. Tongxin Wang: Writing – original draft. Jiaqi Chen: Writing – review & editing, Resources, Data curation. Mingxing Sui: Supervision. Luyao Wang: Software. Xueyu Zhao: Software. Jianqiao Sun: Resources. Yingli Lu: Supervision, Conceptualization.

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This study received financial backing from the Science and Technology Department of Jilin Province, China (Project No.YDZJ202301ZYTS002),Jilin Province Medical and Health Talent Special Project(2024wszx-B16).

3D bioprinting technology holds significant potential for applications in the repair and regeneration of the female reproductive system. However, challenges remain, such as insufficient vascularization in printed tissues and structural and functional discrepancies compared with natural tissues. Addressing these issues requires feasible approaches, including optimizing 3D printing techniques, identifying biocompatible materials suitable for bioprinting, and selecting appropriate cell types and bioactive factors. When integrating 3D bioprinting with stem cell technology for regenerative medicine in the female reproductive system, a key issue is how to induce the directed differentiation of stem cells. 3D bioprinting provides favorable growth conditions for stem cells, facilitating their interaction with the surrounding microenvironment. Furthermore, with advancements in bioprinting materials, such as the development of dECM, it is now possible to create specific physiological and biochemical environments for various cell lines, enriched with growth factors and stem cell niches. This environment supports cell adhesion and proliferation and can, to some extent, induce the directed differentiation of stem cells, thereby promoting tissue formation and regeneration. 3D-bioprinted tumor models for cervical and ovarian cancers have already been successfully employed in studying tumor pathogenesis and drug screening. These 3D-bioprinted tumor models exhibit unique three-dimensional biological characteristics, making them valuable tools for studying tumor biology in a 3D context. However, in vitro tumor experiments often lack the regulatory influences of other human systems. The female reproductive system, consisting of several organs interconnected through complex endocrine pathways and communication mechanisms, poses additional challenges. Consequently, simple in vitro 3D models may yield outcomes that differ from in vivo conditions. "Organ-on-a-chip" technology offers a promising solution by leveraging microfluidics and 3D cell culture techniques to create biologically active organ models on microchips. 3D bioprinting can provide precise spatial structures for three-dimensional cell cultures, while microfluidic systems can better simulate the in vivo microenvironment. Integrating different organs onto a single biochip could enable the creation of more realistic models that closely mimic the physiological or pathological conditions of the female reproductive system, providing a powerful tool for research in this field. Given the current applications of 3D bioprinting in the female reproductive system, there is potential to explore its use in female fertility preservation. Presently, the primary techniques for fertility preservation in women include embryo freezing, oocyte freezing, and ovarian tissue freezing. Among these, embryo freezing is the most effective but is only suitable for married women. For women without a partner or for prepubescent girls, oocyte freezing and ovarian tissue freezing are the only available options. However, oocyte freezing often faces limitations due to the patient's condition, making it difficult to obtain mature oocytes. While immature oocytes can be matured in vitro, the normal development and blastocyst formation rates of embryos derived from in vitro-matured oocytes are lower than those of embryos derived from in vivo-matured oocytes. 3D bioprinting could offer a solution by constructing implants that combine immature oocytes with biomaterials, allowing these oocytes to mature in vivo and thereby increasing the chances of natural conception for such patients. As for ovarian tissue freezing, a common issue is the significant reduction in the number of primordial follicles due to hypoxia during tissue thawing and transplantation. 3D bioprinting can address this by using biomaterials to load ovarian tissue and relevant bioactive factors to construct 3D-structured implants. These implants could facilitate better nutrient exchange between the ovarian tissue and the surrounding environment, reducing hypoxia and improving the survival rate of primordial follicles within the ovarian tissue. 3D bioprinting still faces challenges in fully meeting the demands of the female reproductive system. It is important to recognize that other areas of biological research are also advancing toward the creation of multicellular systems, such as organoids, which address the critical need for microenvironments defined by specific cellular interactions. Moreover,3D bioprinting is confronted with a host of ethical issues, including disputes over the sourcing and utilization of cells, the ethical balance in clinical applications and trials, risks of technological misuse, and challenges regarding intellectual property rights and regulation.Aditionally, bioprinting technology must achieve scalability and integrate innovatively with additional biofabrication methods. However, interdisciplinary collaboration may increase labor costs, and the materials and equipment typically used in 3D bioprinting are often expensive.Consequently, the development of low-cost hardware and improved accessibility are further necessary to advance its application in the field of the female reproductive system.

The health of the female reproductive system is crucial for women's quality of life and population reproduction. Anatomically, the female reproductive system comprises internal and external genitalia. In this article, the term “female reproductive system” primarily refers to the internal genitalia, located within the true pelvis, including the ovaries, fallopian tubes, uterus, cervix, and vagina [ 1 , 2 ]. Diseases, medications, genetics, physical injuries, and other endogenous and exogenous factors can cause damage and dysfunction in the female reproductive system, thereby affecting women's health and fertility [ 3 ].3D bioprinting is an emerging technology that has great potential in tissue and organ construction because of its ability to precisely control the spatial,therefore, therefore,it holds the promise of facilitating breakthroughs in the treatment and research of the female reproductive system. The female reproductive system is a dynamic structure regulated by the hypothalamic-pituitary-gonadal axis, where periodic hormonal changes drive corresponding changes, such as ovulation and the cyclic growth of the endometrium. Therefore, hormone therapy plays a significant role in treating conditions affecting the reproductive system [ 13 ]. For instance, estrogen therapy can promote endometrial repair, thus treating intrauterine adhesions caused by improper curettage [ 14 ]. However, the success of hormone therapy depends on the presence of target cells, as a substantial loss of these cells can limit therapeutic outcomes. Moreover, the reproductive system is susceptible to space-occupying lesions, including tumors, for which surgical intervention is often the primary treatment. In cases of malignancy, additional treatments like radiotherapy and chemotherapy are employed. However, these treatments may damage or remove reproductive organs, impacting both reproductive and endocrine functions [ 15 ]. As a result, there is a pressing need for new treatment strategies to repair damaged reproductive tissues and restore reproductive capacity. The rapid development of regenerative medicine has attracted widespread attention in female reproductive health. Current approaches include autotransplantation of cryopreserved ovarian tissue for women with complete ovarian loss and the use of stem cell perfusion for patients with severe endometrial damage to achieve endometrial repair and regeneration [ 16 ]. However, these strategies lack a three-dimensional structural framework for functional cells, resulting in significant cell loss and challenges in establishing long-term engraftment in the body [ [17] , [18] , [19] ]. 3D bioprinting technology can address these limitations by depositing bioinks into high-resolution scaffolds that act as temporary extracellular matrices (ECMs). This 3D structure provides a microenvironment that supports the growth and proliferation of functional cells, restricts their outward migration, and sustains therapeutic effects within the body [ 20 ]. Thus, 3D bioprinting holds broad application potential in the regenerative medicine of the female reproductive system. Currently, research on the female reproductive system's pathology and physiology relies mainly on animal and cell studies. However, genetic differences and varying metabolic pathways limit animal models' ability to replicate specific human biological processes. For instance, less than 8 % of cancer research findings from animal models advance to clinical trials [ 21 ]. Additionally, ethical concerns restrict the use of animal models. While human cells are preferred in research, traditional 2D cell cultures cannot accurately mimic the human microenvironment, such as growth conditions, cellular signaling, and interactions with neighboring cells or ECM [ 22 ]. The application of 3D bioprinting in creating 3D in vitro models offers a promising approach for studying the female reproductive system. Compared with 2D systems, 3D models demonstrate notable differences in morphology, cell vitality, proliferation, differentiation, and gene expression profiles, providing conditions closer to in vivo [ 23 ]. In studying reproductive system tumors, traditional cell culture models often cannot simulate the later stages of tumor development due to limitations in constructing complex structures and vascular networks. 3D bioprinting technology can accurately control the composition and spatial distribution of tumor-related cells and ECM components, enabling high-resolution and high-throughput creation of tumor models with complex, multi-scale structures, multiple biomaterials, and vascular networks. This is crucial for effective, patient-specific drug screening and biomedical research in reproductive system tumors. For example, cervical tumor spheroid models produced via 3D bioprinting are used to examine cell proliferation, matrix metalloproteinases (MMPs), and the response to paclitaxel treatment. In these models, HeLa cells exhibit increased proliferation, enhanced MMP protein expression, and greater resistance to paclitaxel than traditional 2D culture models [ 4 ]. Therefore, 3D bioprinting presents valuable opportunities for constructing in vitro models of the female reproductive system, particularly for tumor modeling. In this review, we summarize the methods and materials in 3D bioprinting relevant to the female reproductive system and discuss the current applications of 3D bioprinting in tissue regeneration and in vitro model development. Finally, we address challenges and potential solutions for the future application of 3D bioprinting technology in the female reproductive system ( Fig. 1 ). Fig. 1 The main steps of 3D printing of the female reproductive system and its main applications in the female reproductive system. Fig. 1 The main steps of 3D printing of the female reproductive system and its main applications in the female reproductive system.

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