Applications and challenges of 3D printing in female reproductive system research.

The immense versatility and prospects of 3D printing have attracted the attention of researchers in different areas of reproductive biology and medicine. 3D-printed scaffolds with highly defined and detailed designs can recapitulate the complex interaction of different cell types in the reproductive organs. Although tissue engineering reproductive organs by 3D-printed scaffolds is a very attractive and promising topic, it is still in its initial stages, and 3D bioprinting possesses massive potential for future state-of-the-art reconstructive studies. In addition, drug delivery devices can be designed by 3D printing with outstanding precision over the detailed features and the capabilities of different active pharmaceutical agents’ inclusion. These devices have been used for a broad spectrum of reproductive complications, and many future ones will be introduced with novel advancements in 3D printing technology. Furthermore, 3D-printed structures have been utilized as suitable matrices for modeling reproductive diseases, which provided a valuable platform for in-depth study of reproductive complications. In conclusion, the applications of 3D-printed structures in reproductive science are a fast-growing area, and future developments in new printing technologies and ink biomaterials will offer innovative avenues for 3D printing applications in reproductive science.

The female reproductive system comprises several essential organs, including the ovaries, fallopian tubes, cervix, vagina, and uterus. Each of these organs serves a distinct purpose, and proper functioning of each is crucial for successful human reproduction [ 1 , 2 ]. The fallopian tubes serve as the link between the uterus and the ovary. This region is where fertilization occurs, making it a critical part of the reproductive process. Meanwhile, one end of the cervix connects to the uterus, while the other end leads to the vagina [ 3 ]. The precise functioning of this organ system is vital, and the disruption of a part of it affects the health of women. Within the domain of female reproductive health, one of the primary focal points of regenerative medicine is infertility, a multifaceted condition influenced by numerous factors. Diseases of the female reproductive system, including uterine, cervical, and ovarian cancers, disrupt the proper function of the reproductive system, potentially lead to infertility, and also threaten women’s lives. Additionally, complications such as preeclampsia, endometriosis, stress urinary incontinence, intrauterine adhesions, and pelvic organ prolapse can manifest during pregnancy or after childbirth, markedly diminishing one’s quality of life. Infections resulting from bacteria, fungi, and viruses can lead to conditions like bacterial vaginosis, vulvovaginal candidiasis, and genital herpes in women. Moreover, both congenital and acquired factors can influence the health of the female reproductive system [ 4 ]. Despite the advances in reproductive medicine and reconstructive surgery, the demand for restoring the function and compensating the loss of female reproductive tissues and organs has been on the rise [ 5 ]. The integration of innovative engineering techniques into prevention and treatment methods is progressing, making a significant shift in the field. The study of the human reproductive organs demands a multidisciplinary approach, a need that persists until the advancement of biomedical engineering. This field relies on the development of methods and biomaterials to restore, maintain, and enhance tissue functions [ 6 ]. One of the most recent and remarkable advancements in this field is the integration of three-dimensional (3D) printing within the field of bioengineering. 3D printing has emerged as a versatile technology, serving various purposes in the fabrication of tissue-engineered scaffolds, devices (drug delivery devices), disease models, implants, and more. Its diverse range of applications, combined with the growing need for innovative platforms for in vitro and in vivo studies, positions 3D printing as a leading technology in the field of biomedical engineering, dedicated to tissue repair and regeneration. Bioprinting, a subset of 3D printing, excels at crafting uniform biomimetic 3D scaffolds with consistent cell distribution, thereby enabling the 3D culture of cells [ 7 , 8 ]. Moreover, the ability to create microstructures with exceptional precision paves the way for the creation of effective drug delivery systems within the complex environment of the body [ 9 ]. Recently, several 3D-printed models for human organs, including skin [ 10 ], bone [ 11 ], and liver [ 12 ], have been developed. In this review, we explored the applications of 3D printing in female reproductive system research. Broadly, we categorized the applications of 3D printing into three distinct areas: tissue engineering, devices (drug delivery devices), and disease models. To begin, we will explain the history, and various techniques used in 3D printing. Then, we will turn our attention to 3D printing research within the field of the female reproductive system. 3D printing, also known as additive manufacturing or rapid prototyping, is a method for creating three-dimensional objects from digital models of various materials [ 13 ]. Figure 1 illustrates the evolution of 3D printing technology over recent decades. The significant advantages of 3D printing technology include a broad spectrum of designable shapes, customization, toxicity prevention, the capacity to construct intricate systems, and swift execution of prototyping. Bioprinting, a specialized field within 3D printing, focuses on the medical field, specifically in the fabrication of tissues and organs [ 14 , 15 ]. Key 3D printing methods include extrusion-based, stereolithography, inkjet, and laser-assisted printing. Biocompatible biomaterials are utilized in various applications, including tissue engineering, blood vessel replacement, dental implants, and medical prostheses [ 16 ]. The future of 3D printing is promising as research continues to expand in organ and tissue printing technology. 3D printing technology has evolved to meet the demands of various industries, including biomedicine. Researchers utilize it for implantable device prototypes, tissue engineering, organ repair, and the customization of pharmaceutical products [ 16 , 17 ]. It also helps in production of disease models and surgical phantoms for better understanding and treatment of tissue. 3D printing has therapeutic applications in medical engineering, particularly in the female reproductive system [ 18 , 19 ]. In addition, animal models are less reliable for evaluating scaffolds utilized in tissue engineering, necessitating the adoption of new laboratory methods like 3D printing and organ-on-a-chip. These challenges and trends have prompted increased efforts to leverage engineering technologies in the context of female reproductive system research [ 20 , 21 ]. Fig. 1 History of 3D printing method development in regenerative medicine (created on BioRender.com) History of 3D printing method development in regenerative medicine (created on BioRender.com) 3D bioprinting techniques are widely used for tissue engineering scaffolds, drug delivery devices, and disease models. Fused deposition modeling (FDM), semi-solid extrusion (SSE), and direct ink writing (DIW) are three main methods used in the extrusion-based printing technique (Fig. 2 -A). FDM uses thermoplastic polymer filaments, while SSE uses gel or paste in successive layers to construct three-dimensional structures. DIW uses a specific rheology to print ink, limiting material choice and preserving structure in situ [ 18 , 22 – 24 ]. Stereolithography (SLA) is the pioneering commercial 3D printing technique, using a container holding photocurable resin, a laser source, and a motion system (Fig. 2 -B) [ 25 , 26 ]. Laser-assisted bioprinting (LAB) is based on laser-induced forward transport (LIFT) and involves a focusing system, an absorbent layer, a pulsed laser beam, and a substrate for the bioink layer (Fig. 2 -C) [ 27 , 28 ]. Inkjet printing involves applying low-viscosity ink to a substrate with thermal and piezoelectric methods (Fig. 2 -D) [ 29 , 30 ]. Table 1 presents a comprehensive comparison of various 3D printing methods. Fig. 2 3D printing approaches and operational mechanisms: ( A ) Extrusion-based printing utilizes three techniques to sequentially extrude ink layer by layer, forming the structure. ( B ) Stereolithography printing selectively cross-links inks layer by layer using UV or visible light, constructing a 3D model. ( C ) Laser-based printing directs a laser onto an absorbing layer. ( D ) Inkjet-based printing releases fractions of ink molecules through extrusion (created in BioRender.com) Fig. 3 The survival of ovarian follicles relied on multiple contacts facilitated by scaffold pore geometries at 30° and 60° angles. ( A-C ) 3D reconstructions of confocal fluorescence images depicted scaffolds at advancing angles of 30° ( A, D ), 60° ( B, E ), and 90° ( C, F ), with struts around 250 μm and a strut-to-strut distance of ~ 350 μm. ( D-F ) maximum intensity projections illustrated GFP+ follicles in pores after 2 days, revealing a tendency for follicles in 30° and 60° pores to have resided in corners, while those in 90° pores were more likely to have aligned along a single strut. ( G ) survival analysis, based on scaffold geometry, indicated optimal follicle thriving in 30° and 60° scaffolds ( p  = 0.0146). ( H ) percentage distribution of follicles making 1, 2, or 3 scaffold contacts per geometry showed a higher likelihood of 2 or 3 contacts in 30° and 60° scaffolds. Scale bars: ( D-F ) 100 μm. All data is presented as average ± s.e.m. Statistical significance was assessed using one-way anova with Holm-Sidak’s multiple comparisons test (* p  < 0.05; ** p  < 0.005) [ 41 ]. (permission was granted to reuse the figure from Springer Nature) Table 1 Comparison of different parameters of 3D bioprinting methods Extrusion-based printing Stereolithography Laser-assisted printing Inkjet printing Typical Materials Hydrogels, biomolecules, living cells Photo-curable polymer resins Thermoplastic polymer, metal and ceramic Polymers, hydrogels cost Medium Low High Low Cell viability 40–80%  > 85%  > 85%  > 85% Cell density High (cell spheroids) -  < 10 8 cells/ml  < 10 6 cells/ml Resolution 5 mm 50–100 mm  < 500 nm 50 mm Printing speed Slow (10–50 mm/s) Fast (mm 3 /s) Medium (mm/s) Medium (mm/s) Viscosities - 6–30 × 107 mPa/s 1–300 mPa/s 3.5–12 mPas/s 3D printing approaches and operational mechanisms: ( A ) Extrusion-based printing utilizes three techniques to sequentially extrude ink layer by layer, forming the structure. ( B ) Stereolithography printing selectively cross-links inks layer by layer using UV or visible light, constructing a 3D model. ( C ) Laser-based printing directs a laser onto an absorbing layer. ( D ) Inkjet-based printing releases fractions of ink molecules through extrusion (created in BioRender.com) The survival of ovarian follicles relied on multiple contacts facilitated by scaffold pore geometries at 30° and 60° angles. ( A-C ) 3D reconstructions of confocal fluorescence images depicted scaffolds at advancing angles of 30° ( A, D ), 60° ( B, E ), and 90° ( C, F ), with struts around 250 μm and a strut-to-strut distance of ~ 350 μm. ( D-F ) maximum intensity projections illustrated GFP+ follicles in pores after 2 days, revealing a tendency for follicles in 30° and 60° pores to have resided in corners, while those in 90° pores were more likely to have aligned along a single strut. ( G ) survival analysis, based on scaffold geometry, indicated optimal follicle thriving in 30° and 60° scaffolds ( p  = 0.0146). ( H ) percentage distribution of follicles making 1, 2, or 3 scaffold contacts per geometry showed a higher likelihood of 2 or 3 contacts in 30° and 60° scaffolds. Scale bars: ( D-F ) 100 μm. All data is presented as average ± s.e.m. Statistical significance was assessed using one-way anova with Holm-Sidak’s multiple comparisons test (* p  < 0.05; ** p  < 0.005) [ 41 ]. (permission was granted to reuse the figure from Springer Nature) Comparison of different parameters of 3D bioprinting methods While we explore the frontiers of reproductive medicine, the incorporation of cutting-edge technologies, such as tissue engineering and 3D printing, is revolutionizing the landscape. This dynamic synergy holds enormous promise for addressing critical challenges in fertility preservation and assisted reproductive technologies [ 31 ]. Tissue engineering assumes a pivotal role in fabricating biocompatible scaffolds that faithfully mimic the intricate microenvironment of reproductive tissues. These scaffolds provide an optimal substrate for the growth and maturation of ovarian follicles, marking a breakthrough in fertility preservation [ 6 ]. Tissue engineering offers precise control over scaffold geometries, pore sizes, and material properties, enabling the recreation of optimal conditions for follicle survival. A 3D bioprinter can achieve precise control over the mentioned parameters [ 2 ]. The following presents some examples of the integration of tissue engineering and 3D bioprinting in reproductive medicine. In uterine tissue engineering, a 3D bioprinter was used to create customized scaffolds and simulate in vitro models of the myometrium layer, which facilitated the study of the physiology of uterine contraction [ 32 ]. In addition, 3D printing helped to regenerate damaged endometrial tissue [ 33 – 35 ]. Furthermore, 3D printing has played an important role in placental tissue engineering, enabling the construction of complex models to study trophoblast invasion, a key aspect of placental health. These printed models facilitated the exploration of interactions between bioactive agents, cells, and biomaterials and provided information about placental pathology and potential therapeutic strategies [ 36 – 38 ]. The promising results of 3D bioprinting in vaginal tissue engineering promise to restore vaginal function by regenerating its tissue in the future [ 39 ]. Furthermore, the integration of tissue engineering and 3D printing has provided new strategies in the development of in vitro culture systems. Through the use of engineered scaffolds with precisely defined geometries, researchers can establish microenvironments that closely mimic the in vivo conditions essential for the successful development of reproductive tissues. This approach not only advances our comprehension of follicle biology but also offers a platform for exploring innovative strategies in fertility preservation and restoration [ 40 ]. Current treatment methods to address some women’s diseases, including cancer, can hurt the ovary and disrupt its function. In addition, serious disorders such as puberty failure, premature menopause, and infertility, for which current treatment methods do not provide long-term solutions, require novel innovative therapeutic methods. In recent studies, engineered ovaries using a 3D printer have provided promising results for the long-term treatment of ovarian diseases. In this regard, Laronda et al. created a murine bioprosthetic ovary using a 3D bioprinter. For this purpose, they fabricated tortuous porous gelatin scaffolds seeded with murine follicles using a 3D printing technique based on extrusion. They investigated how the pores’ geometry would affect ovarian follicles’ survival. They evaluated the print angles of 30, 60, and 90 degrees in this regard (Fig. 3 –A, B, and C). The results indicated that the scaffolds with printing angles of 30 and 60 degrees form corners that surround the follicles from several sides. At the same time, the 90-degree printing angle limited the interaction between the scaffold and the follicle (Fig. 3 –D, E, and F). The survival results indicated that optimal follicle thriving occurs in scaffolds with 30° and 60° angles (Fig. 3 –G). Furthermore, the results indicated that the number of follicles that have two or three contacts with the scaffold are at 30° and 60° angles (Fig. 3 –H). Ovarian function was restored by implanting highly vascularized follicle-seeded scaffolds into surgically sterilized mice. Also, mouse pups were born through natural mating and grew by consuming the mother’s milk [ 41 ]. Premature ovarian insufficiency is a problem that may occur due to the gonadotoxic effects of some cancers. Using safe transplants for long-term hormone restoration can be a promising solution for this disease. To achieve this goal, Henning et al. investigated the contribution of the ovarian microenvironment to its physical and biochemical properties to inform bioprosthetic ovary scaffolds. For this purpose, they created constructs using decellularized bovine ovarian cortex and medulla extracellular matrix (ECM) hydrogels and evaluated them in vitro as bioprosthetic ovary scaffolds. They observed a stiffness gradient in the cow’s ovary, which the printed scaffolds also maintained. This finding was due to the presence of matrisome proteins. The removal of EMILIN1 and AGRN (ECM glycoproteins) did not alter the stiffness of the printed gels or scaffolds. Researchers found these materials to be comparable to unfilled hydrogels and inks [ 42 ]. In another study, Wu et al. used the extrusion technique to produce an artificial 3D ovary seeded with exogenous follicles. They used GelMA as a bioink. The results indicated that the GelMA-based 3D printing system created a favorable environment for ovarian follicles, promoting their growth and ovulation within the scaffolds. However, the viability of primary ovarian somatic cells was lower than that of commercial cell lines because extrusion-based 3D culture fabrication was not suitable for primary ovarian cells [ 43 ]. Li et al. prepared a bioink based on decellularized pig ovarian tissue, gelatin, and alginate. After loading adipose-derived stem cells (ADSCs) into the bioink, they used a 3D bioprinter to create scaffolds for the engineering of ovarian tissue. Furthermore, they used premature ovarian insufficiency rats as an in vivo model to evaluate the fabricated scaffolds. Overall, their accomplishments include notable enhancements in ovarian function, marked by significant improvements in follicle counts, granulosa cell proliferation, neo-angiogenesis, and hormone levels [ 44 ]. In a similar study, Zheng et al. created structures for mouse ovarian failure correction by employing hydrogels based on swine ovarian decellularized extracellular matrix (dECM), seaweed gelatin, and sodium alginate. Experimental in vivo groups included 3D-printed scaffolds without cells, 3D-printed scaffolds with primary ovarian cells (POCs), and bioink with POCs without printing, which were compared to the control group (ovariectomized mice without further treatment) and sham group (non-ovariectomized). They confirmed the secretion of sex hormones and observed that scaffolds containing POCs enhanced neoangiogenesis, cell proliferation, and survival. Furthermore, the expression of germ cell genes such as ER-α, PR, inhibin-α, and FSHR was higher in the printed groups in which POCs were encapsulated than in the non-printed groups [ 45 ]. Uterine contraction is the responsibility of the uterine myometrium layer, which facilitates some functions, such as the menstrual cycle, transport of sperm and embryos, pregnancy, and parturition. Disruption of uterine contraction function can lead to an irregular menstrual cycle, abnormal implantation, preterm labor, premature birth, and infertility. We need a more profound understanding of the physiology of uterine contraction to prevent and treat this problem. For this purpose, Souza et al. attempted to evaluate the contraction by simulating a 3D in vitro model of the myometrium layer of the human uterus using magnetic 3D bioprinting. They used magnetic 3D bioprinting to simulate a 3D in vitro model of the myometrium layer. Using magnetic technology, they printed human myometrial cells to create hollow rings resembling the cross-section of the uterus, a hollow organ. They examined the contraction of the created model over time as a function of tocolytic agents. They observed the contraction of cell rings printed with commercial smooth muscle cells and patient-derived human uterine myometrial cells immediately after printing. Furthermore, the results showed that indomethacin and nifedipine (tocolytic factors) have a calming effect on simulated uterine contractions and almost stop them [ 32 ]. Other potential issues for the uterus include irreversible endometrial damage, which can result from uterine curettage or inflammation and may lead to infertility in women. To regenerate the damaged endometrial tissue, Ji et al. used microextrusion 3D printing to fabricate hydrogel scaffolds based on sodium alginate and gelatin loaded with human-induced pluripotent stem cell-derived mesenchymal stem cells (hiMSC). The results indicated that printed hydrogel scaffolds provided a suitable environment for cells in vitro and increased the survival time of transplanted hiMSCs in Sprague-Dawley rats. The results showed that, although the constructed scaffold could not completely restore the structure and function of the endometrium, it was able to restore endometrial histomorphology and the regeneration of endometrial and endothelial cells. It also improved endometrial receptivity functional indicators and restored embryo implantation and pregnancy maintenance functions [ 33 ]. Using the same bio-ink, Wen et al. put a sustained-release PLGA microsphere containing granulocyte colony-stimulating factor (G-CSF) into a hydrogel composition and made scaffolds with an extrusion-based 3D printer for endometrial regeneration. They observed that the use of microspheres increased the local concentration of G-CSF. An in vivo IUA (intrauterine adhesion) rat model showed that a microsphere-loaded scaffold improved local endometrial regeneration, the formation of endometrial cell layers, vascular regeneration, endometrial receptivity, and the pregnancy function of the damaged endometrium. Endometrial tissue fibrosis was also effectively suppressed [ 34 ]. Nie et al. employed a 3D extrusion-based bioprinting technique to create double-layered scaffolds for endometrium function regeneration, utilizing a sodium alginate-hyaluronic acid hydrogel. The bottom layer of the scaffold consisted of a grid-like microstructure loaded with endometrial stromal cells, which was covered by a monolayer of endometrial epithelial cells. Fig. 4 –A, B, C, and D display the microstructure, cell distribution, attachment, and growth. We successfully created a bilayer endometrial construct and maintained cell viability through the 3D bio-printing process (Fig. 4 –E). Using a rat model with partial uterine excision, the bilayer endometrial construct restored endometrial structure and improved reproductive outcomes post-implantation [ 35 ]. Fig. 4 Evaluation of primary endometrial epithelial cells (EECs), endometrial stromal cells (ESCs), and the 3D bio-printed bilayer endometrial construct (ec). ( A ) Immunofluorescence (if) staining was used to assess the expression of pan-cytokeratin, an epithelial marker in red, and fibronectin (fn), a mesenchymal marker in green, in primary EECs and ESCs. ( B ) Light microscope images show the microstructures of the bio-printed scaffold. ( C ) Cell distribution in the hydrogel was observed, with EEC and ESC labeled by DiO and DiI, respectively. ( D ) Sem images were taken after 24 hours to observe eec (upper) and esc (lower) attachment and growth in the bio-printed scaffold. ( E ) Minimal cell death was demonstrated by confocal scanning fluorescence microscopy images of ECSs in bioprinted scaffolds via live-dead assay (calcein AM and PI) within 48 hours after printing. Scale bars: ( A ) 100 μm, ( B,C ) 500 μm, ( D ) 5 μm, ( E ) (blue = 500 μm, white = 200 μm) [ 35 ] (permission was granted to reuse the figure from Elsevier) Evaluation of primary endometrial epithelial cells (EECs), endometrial stromal cells (ESCs), and the 3D bio-printed bilayer endometrial construct (ec). ( A ) Immunofluorescence (if) staining was used to assess the expression of pan-cytokeratin, an epithelial marker in red, and fibronectin (fn), a mesenchymal marker in green, in primary EECs and ESCs. ( B ) Light microscope images show the microstructures of the bio-printed scaffold. ( C ) Cell distribution in the hydrogel was observed, with EEC and ESC labeled by DiO and DiI, respectively. ( D ) Sem images were taken after 24 hours to observe eec (upper) and esc (lower) attachment and growth in the bio-printed scaffold. ( E ) Minimal cell death was demonstrated by confocal scanning fluorescence microscopy images of ECSs in bioprinted scaffolds via live-dead assay (calcein AM and PI) within 48 hours after printing. Scale bars: ( A ) 100 μm, ( B,C ) 500 μm, ( D ) 5 μm, ( E ) (blue = 500 μm, white = 200 μm) [ 35 ] (permission was granted to reuse the figure from Elsevier) Inadequate trophoblast invasion causes poor placentation, which has complications such as reduced placental perfusion, oxidative stress, and the production of inflammatory cytokines. Subsequently, this problem may result in preeclampsia, which is the main cause of maternal and perinatal morbidity and mortality [ 36 , 46 ]. The lack of effective experimental models has led to a poor understanding of the pathology of this problem. In this regard, Kuo et al. used bioprinting and shear wave elastography to make three-layer cylindrical models of the placenta based on GelMA. The outer layer of the scaffold contained fibronectin along with BeWo cells (a trophoblast cell line) and human mesenchymal stem cells (hMSCs); the middle layer contained fibronectin; and the inner layer contained EGF. The release of EGF created a concentration gradient that caused cells to migrate toward the inner layers, prompting the researchers to devise a model for studying and quantifying cell migration. They observed that there is a positive correlation between trophoblast and hMSC migration rates and EGF concentration. Furthermore, live/dead staining results indicated that BeWo cells and hMSCs in the printed scaffold had more than 90% survival after 8 days of culture. In general, they showed that by using an ex vivo placental model, the interaction between bioactive agents, cells, and biomaterials can be investigated and therapeutic methods can be provided [ 36 ]. In a similar study, the phenomenon of trophoblast cell invasion was evaluated using a 3D bioprinted model based on GelMA. For this purpose, three-layer scaffolds with a multi-strip model were printed with the presence of EGF in the central layer and the HTR8/SVneo cell line in the outer layer. GelMA composed the inner layer. Due to the limitations of the 3D model (multi-ring model), they developed a 2D multi-strip model to investigate cell movement in the presence or absence of EGF. The results showed that in the multi-ring model, the cells move at a rate of 85 ± 33 µm/day (with an EGF gradient of 16 µM), while when the model was covered with a layer of GelMA (for limiting the cells in the 3D environment), this parameter decreased to 13 ± 5 µm/day. In cases where the model was not covered by GelMA, there was no significant difference in cell front movement between the control and EGF-stimulated rates. But when the model was covered with GelMa (3D multi-strip model), EGF-stimulated cells showed an invasion rate of 21 ± 3 µm/day compared to 4 ± 5 µm/day for non-EGF stimulation [ 37 ]. Kuo et al. developed a GelMa-based bilayer tubular model with the help of a 3D bioprinter and placed it in a perfusion bioreactor system for dynamic culture. The scaffold was printed in such a way that the inner layer of the tube contains human umbilical vein endothelial cells (HUVECs) and its outer layer contains trophoblasts. They observed that dynamic culture in the perfusion bioreactor led to increased endothelial cell response by encouraging network formation and expression of angiogenic markers CD31, MMP2, MMP9, and VEGFA. They also observed that trophoblasts reduced the angiogenic response as a result of reducing network formation and motility rates and inducing apoptosis in endothelial cells. Furthermore, the results indicated that HUVECs inhibited the trophoblast invasion rate [ 38 ]. Several factors can be involved in the loss of vaginal function, and patients with this condition endure severe physical and psychological pain that requires the reconstruction of the vaginal tissue. For this purpose, the use of 3D bioprinters for vaginal tissue engineering is very promising. Hou et al. fabricated scaffolds for vaginal tissue engineering by using biological inks based on pig acellular vagina matrix, sodium alginate, and gelatin. The bioink was loaded with bone marrow mesenchymal stem cells (BMSCs). Furthermore, they evaluated the engineered vagina in vivo by implanting the scaffold in rats subcutaneously. They reported that engineered vaginas encapsulating BMSCs had significant vascularization and epithelization. Furthermore, BMSCs can acquire the phenotype of vaginal epithelial cells and endothelial-like cells [ 39 ]. Despite the satisfactory outcomes of vaginal tissue engineering using 3D bioprinters, this field still requires further studies. Table 2 summarizes all the studies on the use of 3D printing in the tissue engineering field. Table 2 Tissue engineering of reproductive organs using 3D printing technologies Ref. 3D Printing method Related disease/target tissue Ink(s) Type of study (Model) Cell components (Origin) API(s) Outcome Laronda et al. [ 41 ] Extrusion -/ovary Gelatin In vitro, in vivo (mice) Follicles (isolated from female mice) - A 3D bioprinted ovary restored ovarian function and enabled natural mating in mice. Henning & Laronda [ 42 ] FRESH Premature ovarian insufficiency/ovary Cortex and medulla-derived dECM inks were mixed 1:1 with high concentration COL1 In vitro - - ECM hydrogel stiffness matched the cow’s ovary. Wu et al. [ 43 ] Extrusion -/ovary GelMA In vitro COV434, KGN, ID8, ovarian somatic cells (4-week-old female C57BL/6J mice), and follicles (3-week-old female C57BL/6J mice) - Ovarian follicle growth and ovulation in the scaffold were promoted. Li et al. [ 44 ] Extrusion Premature ovarian insufficiency/ovary dECM (porcine ovary tissue)/sodium alginate/gelatin In vitro, in vivo (rat) ADSCs - Ovarian function was enhanced with improvements in follicle counts, granulosa cell proliferation, neo-angiogenesis, and hormone levels. Zheng et al. [ 45 ] Extrusion Ovarian failure/ovary dECM (swine ovarian tissues)/gelatin/sodium alginate In vitro, in vivo (mice) Primary ovarian cells (4-week-old female Kunming mice) - Scaffold-incorporated POCs enhanced hormonal secretion, neoangiogenesis, and cell survival. Souza et al. [ 32 ] Magnetic 3D bioprinting Uterine contractility disorders/uterus SMCs In vitro HUtSMCs (two cell samples: commercial sample and isolated from uterine biopsies) Indomethacin, Nifedipine, Ibuprofen Uterine contractions in created hollow rings were simulated, and tocolytic agents almost stopped the contractions. Ji et al. [ 33 ] Microextrusion 3D printing Repair of the endometrium/uterus Gelatin/sodium alginate In vitro, in vivo (rat) hiMSCs (derived from the 6F/BM1-4C system) - Although the scaffold did not fully restore the endometrial structure, it successfully restored embryo implantation and pregnancy maintenance functions. Wen et al. [ 34 ] Extrusion Intrauterine adhesions/uterus Gelatin/sodium alginate In vitro, in vivo (rat) rESCs (isolated from female rat uterine tissues) G-CSF-SRM (Recombinant human G-CSF+dextran+polyethylene glycol) Endometrial regeneration, vascular regeneration, endometrial receptivity, and pregnancy function were improved. Nie et al. [ 35 ] 3D extrusion-based bioprinting (EBB) Treatment for severe endometrial injury/uterus Sodium alginate/hyaluronic acid In vitro, in vivo (rat) EECs and ESCs were isolated from the uterine horns of neonatal rats - Endometrial structure was restored, and reproductive outcomes post-implantation were improved. Kuo et al. [ 36 ] Extrusion Preeclampsia/placenta GelMA/EGF, GelMA/fibronectin In vitro, ex vivo (human placental tissue) BeWo cells, hMSCs - Creating an EGF gradient caused cells to migrate to the inner layers. BeWo cells and hMSCs survived more than 90%. Ding et al. [ 37 ] Microextrusion-based bioprinting (micro-EBB) -/placenta GelMA/lithium phenyl-2,4,6-trimethylbenzoylphosphinate/fibronectin/EGF In vitro HTR-8/SVneo (HTR-8) cells When the 2D MSM was covered with GelMA (3D MSM), the rate of cell invasion increased. Kuo et al. [ 38 ] Extrusion Preeclampsia/placenta GelMA/fibronectin In vitro HUVECs and HTR8 cells - Dynamic culture increased endothelial cell response, trophoblasts reduced the angiogenic response, and HUVECs inhibited the trophoblast invasion rate. Hou et al. [ 39 ] Extrusion Repair of the vagina tissue/vagina AVM from pig/gelatin/sodium alginate In vitro, in vivo (rat) BMSCs (4-week-old male SD rats) - Engineered vaginas were vascularized and epithelialized, and BMSCs acquired the phenotype of vaginal epithelial and endothelial cells. API, active pharmaceutical ingredients; dECM, decellularized extracellular matrix; COL, collagen; ECM, extracellular matrix; GelMA, gelatin-methacryloyl; ADSCs, adipose-derived stem cells; POCs, primary ovarian cells; SMCs, smooth muscle cells; HUtSMCs, human uterine smooth muscle cells; HiMSCs, human induced pluripotent stem cell-derived mesenchymal stem cells; rESCs, rat endometrial stromal cells; G-CSF-SRM, granulocyte colony-stimulating factor-sustained release microsphere; EECs, primary endometrial epithelial cells; ESCs, endometrial stromal cells; EGF, epidermal growth factor; hMSCs, human mesenchymal stem cells; MSM, multi-strip model; HUVECs, human umbilical vein endothelial cells; AVM, acellular vagina matrix; BMSCs, bone marrow stromal cells Tissue engineering of reproductive organs using 3D printing technologies API, active pharmaceutical ingredients; dECM, decellularized extracellular matrix; COL, collagen; ECM, extracellular matrix; GelMA, gelatin-methacryloyl; ADSCs, adipose-derived stem cells; POCs, primary ovarian cells; SMCs, smooth muscle cells; HUtSMCs, human uterine smooth muscle cells; HiMSCs, human induced pluripotent stem cell-derived mesenchymal stem cells; rESCs, rat endometrial stromal cells; G-CSF-SRM, granulocyte colony-stimulating factor-sustained release microsphere; EECs, primary endometrial epithelial cells; ESCs, endometrial stromal cells; EGF, epidermal growth factor; hMSCs, human mesenchymal stem cells; MSM, multi-strip model; HUVECs, human umbilical vein endothelial cells; AVM, acellular vagina matrix; BMSCs, bone marrow stromal cells Gynecological diseases, such as endometriosis and uterine cancer, affect a significant number of women worldwide and present challenges for both diagnosis and treatment. In this regard, the design of a suitable drug delivery system that supports the sustained and controlled release of active pharmaceutical agents and ingredients seems crucial. Recently, 3D printing and bioprinting technology have shown enormous potential in the fabrication of insertable and implantable drug delivery systems such as vaginal pessaries, intravaginal rings (IVRs), intrauterine devices (IUDs), and suppositories with specific and predetermined raw materials, active agents, and geometrically complex patterns or structures [ 47 – 49 ]. Cervical, ovarian, and uterine cancers are among the most widespread gynecological cancers [ 50 ]. One of the leading causes of cancer death among females, cervical cancer, which is mostly caused by long-term infection with specific types of human papillomavirus (HPV), is proposed to be treated by administering therapeutic agents to the mucosal vaginal membrane locally [ 51 ]. Here, 3D printing could offer a suitable opportunity to create patient-specific systems for improved geometrical adjustment. A hydrogel-based localized drug delivery system of gold nanoparticles (AuNPs)-loaded Pluronic F127/alginic acid sodium salt in the lens-shaped geometry was successfully printed using an extrusion-based 3D printing system by Kiseleva and co-workers [ 52 ]. The authors reported a cumulative release of approximately 80% of the encapsulated AuNPs within almost 48 hours, a maximum swelling ratio of 450% in 2 hours, and suitable biocompatibility of the 3D-printed formulations on HeLa, CRL 2616, and BT-474 cells. Positron emission tomography showed that, following a permeation lag time of approximately 3.3 hours, nearly 47% of the administered nanoparticles accumulated in the vaginal membranes within 42 hours, indicating effective tissue retention and potential for sustained drug delivery. In addition, the geometrical precision of printed formulations for translational applications was measured using magnetic resonance imaging (MRI) results, and average percentage deviations of 0.5 and 0.75% (for diameter and height) in lens-shaped scaffolds and 2.4 and 2.1% (for diameter and height) in hollow tube-shaped geometries were calculated. In another similar study, a product consisting of both cyclodextrin inclusion complexes of paclitaxel (as an anticancer agent) and cidofovir encapsulated in polycaprolactone NPs (as an antiviral agent) was printed onto hydroxypropyl cellulose (HPC) substrate using an inkjet printing apparatus for cervical local cervical cancer [ 53 ]. After successfully synthesizing ink and substrate, the cytocompatibility of drug-free and anticancer activity of drug-loaded formulations was confirmed by L929 mouse fibroblast cells and human cervical adenocarcinoma cells, respectively. Their findings demonstrated that paclitaxel- and cidofovir-containing ink and film formulations have a higher anticancer potency than their solutions. Uterine cancer is among the five most common cancers in women, according to the American Cancer Society [ 54 ]. The diverse responses of patients to standard and predetermined protocols in chemotherapy have inhibited the achievement of a suitable treatment for this cancer. Varan et al. [ 55 ], in another study using the hot-melt extrusion method, introduced a dual anticancer drug-loaded PCL filament with sustained release of paclitaxel and carboplatin (up to 10 days) by FDM 3D printing. Their findings demonstrated a decrease in cell viability of HEC-1B human endometrial adenocarcinoma cells by over 60% after treatment with paclitaxel-carboplatin PCL filaments. Genina [ 56 ] and Hollander’s [ 57 ] team reported two controlled releases of indomethacin intrauterine systems (IUSs) using FDM technology with PCL and EVA. They reported the 30-day cumulative release of ~99%, ~87%, and ~67% in PCL-IUSs with 5, 15, and 30% of drug content and 31 and 21% in EVA-IUSs with 5 and 15% drug content, respectively. Another gynecological cancer with a high mortality rate and increasing annual incidence is ovarian cancer (OC) [ 58 ]. Cisplatin is a well-known anticancer drug that interferes with the mitotic division of cancer cells by cross-linking their DNA [ 59 ]. However, its inevitable toxic consequences on other healthy tissues and increasing therapeutic indices make selective delivery of cisplatin an appealing topic in drug delivery. Wang and co-workers [ 60 ] successfully fabricated a cisplatin-poly(lactic-co-glycolic acid) (CDDP-PLGA) compound stent using 3D printing. They acknowledged that CDDP-PLGA significantly increased the efficacy in terms of cytotoxicity as well as changes in apoptosis in SKOV3 and A2780 cell lines at protein and gene expression levels in vitro. For in vivo studies, athymic BALB/c nude female mice were used to imitate tumor growth by subcutaneously implanting SKOV3 into the back and neck. On the 40 th day, the side effects of CDDP and weight loss in the groups treated with CDDP-PLGA were less severe than those observed in the groups treated with CDDP alone. In another similar study performed by Cho and co-workers [ 61 ], Poloxamer 407 nanogel discs (12 mm diameter and 1 mm in thickness) containing paclitaxel and rapamycin were successfully printed using an extrusion-based 3D printing system, and their effectiveness in peritoneal drug delivery and postsurgical peritoneal adhesions was evaluated both in vitro and in vivo. The in vitro results revealed the release of 60% of paclitaxel and 82% of rapamycin at a 24-hour time point. Furthermore, an intermediate dose of paclitaxel and rapamycin is necessary to treat paclitaxel-resistant ES-luc cancer cells. However, a high dose of both drugs is synergistically more effective at treating ES-2-luc cancer cells. In addition, in vivo results indicated that discs containing drugs prevented post-surgical peritoneal adhesion and increased the survival rate in ovarian cancer-bearing xenograft mice. Estrogen controls the growth of cells in the endometrial lining, while progesterone antagonizes it. Endometrial cancer occurs when estrogen and progesterone balances are disrupted inside the uterus, which finally leads to the formation of malignant tumors [ 62 ]. Salmoria and co-workers [ 63 ] used a selective laser sintering (SLS) method and commercial polyethylene to develop an intrauterine implant loaded with progesterone and fluorouracil to treat both endometrial and ovarian cancer. The IUDs prepared using 5W laser power showed better flexural modulus and burst release of fluorouracil, which is crucial for improving the bioavailability of drugs just after implantation. In addition, linear and controlled release of progesterone (~18% in 38 days) was reported, which is effective for sustained levels of the hormone therapy agent in the region of the tumor. Far from the cancers described above, other gynecological problems caused by bacterial, fungal, and viral infections, as well as endometriosis and uterine fibroids, are among the most neglected fields of study in which 3D printing and personalized medicine approaches could enable clinicians to control their outbreak. Vulvovaginal candidiasis is a fungal infection caused by Candida albicans, Candida sp., or yeast pathogens. The typical treatment for this infection is conventional formulations, which include imidazole drugs [ 64 ]. An intravaginal ring (IVR) made up of clotrimazole (CTZ)-loaded polyurethane with two different drug contents (2 and 10% w/w) was fabricated by Tiboni et al. [ 65 ] using the hot melt extrusion method (HME) to treat vulvovaginal candidiasis. As shown in Fig. 5 –A, the agar-diffusion test demonstrated that the plate with IVR containing 10% drug had a larger growth inhibition area compared to IVR with 2 and 0% drug. Furthermore, drug release results showed sustained release over 7 days in both 50% ethanol and vaginal fluid simulant media. These results were also in satisfactory compliance with the Higuchi equation of release. In addition, the viability reduction of C. albicans was highly significant in vaginal fluid simulant media containing 10% CTZ-IVR compared to 0% CTZ-IVR, according to the time-kill experiment, which suggests such IVRs are a therapeutic candidate for the treatment of vulvovaginal candidiasis. In another similar study by Moroni and co-workers [ 66 ], filaments made up of bifonazole (BFZ) and clotrimazole (CTZ)-loaded ethylene-vinyl acetate copolymers were used to prepare IVRs by an FDM 3D printer device. As demonstrated in Fig. 5 –B, the anti-candida activity of IVRs (10% BFZ and CTZ) was significantly higher than that of blank IVRs after 24 hours. Comparing two drug-loaded IVRs, the area of growth inhibition in the CTZ-loaded IVR was remarkably higher than the BFZ-loaded one, which was proposed to be relevant with better release of CTZ. Time-kill assay results also indicated that the decrease in viability of C. albicans in the presence of CTZ-loaded IVR was 32 times higher than BFZ-loaded IVR after 24 hours. Furthermore, Patel et al. [ 67 ] used an FDM printer to fabricate an itraconazole (ITZ)-loaded dumbbell-shaped insert using commercial polyvinyl alcohol (PVA) filaments; also, the polycaprolactone and Tween 80 coatings were applied to control the release rate and form adequate pores. Their outcomes demonstrated the release of 90% ITZ in 48 hours and the degradation of 80% of the insert matrix in 4 days. They also concluded that the formation of pores on the surface of the coated matrix, in comparison with the non-coated matrix, could contribute to the controlled release of the drug from the insert. Fig. 5 Preliminary antifungal activity of prepared IVRs. ( A ) IVR: polyurethane containing 0, 2, and 10% ctz [ 65 ], ( B ) IVR: ethylene-vinyl acetate copolymer containing 0, 10% BFZ, and 10% ctz [ 66 ] (permission was granted to reuse the figures from Elsevier) Preliminary antifungal activity of prepared IVRs. ( A ) IVR: polyurethane containing 0, 2, and 10% ctz [ 65 ], ( B ) IVR: ethylene-vinyl acetate copolymer containing 0, 10% BFZ, and 10% ctz [ 66 ] (permission was granted to reuse the figures from Elsevier) The change in vaginal microbiota from predominant and healthy lactobacilli to facultative anaerobic bacteria such as Gardnerella, Bacteroides, and Anaerococcus is the main cause of bacterial vaginosis (BV), for which the common and available treatment is considered antibiotics in typical oral dosage forms [ 68 , 69 ]. Intravaginal devices can provide a sustained and prolonged release of such antibiotics, and 3D printing technology could effectively address the fabrication challenges. Semi-solid extrusion (SSE) 3D printing technology was used to print metronidazole (MTZ)-loaded polycaprolactone (PCL)/copolymer of methyl vinyl ether and maleic anhydride intravaginal devices in the shapes of mesh and disc by Utomo et al. [ 70 ]. They acknowledged that the disc formulations contained 60/40 and 70/30 ratios of high molecular weight PCL to low molecular weight PCL and were able to sustain the release of the MTZ in 72 h. Furthermore, the release of MTZ in discs containing 50/50 PCL content was extended up to 9 days. They also confirmed that the PCL content ratio did not significantly affect the release of MTZ in printed meshes. Finally, printed discs containing 5% w/w MTZ successfully inhibited the growth of Gardnerella vaginalis, which suggests such devices as alternatives for classic oral antibiotic administrations for the treatment of BV. Besides typical antibiotic-based treatments, probiotics, as living microorganisms, can provide several benefits to patients by providing an antimicrobial compound, lactic acid, and competing with undesired anaerobic bacteria [ 71 ]. Kyser and co-workers [ 72 ], using an Allevi 3 Bioprinter, printed several Lactobacillus crispatus-containing scaffolds with different weight-to-volume ratios of gelatin/alginate in the geometry of IVRs. Their findings suggested a 10:2 (w/v) gelatin/alginate formulation with dual crosslinking by genipin and CaCl 2 as the best formulation in terms of structural stability, minimal mass loss and swelling, post-printing viability, and sustained release of microorganisms over 28 days. Moreover, TPU (thermoplastic polyurethane) and silicon-based materials are other promising substances whose 3D printed devices in the shapes of IVRs and cylinders have demonstrated satisfactory mechanical, imidazole release, and reductive bacterial activity properties and could be used as ink for drug delivery to the female reproductive tract in the treatment of BV [ 73 – 75 ]. Herpes simplex virus (HSV), a DNA virus primarily responsible for increasing the risk of HIV infection and neonatal herpes, is another pathogen for which 3D printing technology can address the typical drawbacks of oral drug administration and low bioavailability, suggesting better treatment options [ 76 ]. Acyclovir is the most commonly administered drug for the treatment of HSV, and acyclovir-incorporated ethylene vinyl acetate (EVA) devices with different 0, 10, and 20 wt.% drug content were fabricated by a fused filament fabrication (FFF) printing device in two geometries: intrauterine device and intravaginal ring by de Carvalho Rodrigues et al. [ 77 ]. Their release results demonstrated a burst release in the first 24 hours, followed by an 80-day slow and sustained release. In addition, in vitro biological assays showed a 99% decrease in HSV-1 replication, which suggested 3D printing as a promising solution for long-term treatment. To combat endometriosis, Teworte et al. [ 78 ] evaluated vaginal ovules manufactured by semisolid extrusion 3D printing using alginate and hydroxypropyl methylcellulose (HPMC) containing pirfenidone. To make a comprehensive comparison, ovules with two excipients of Macrogol and Witepsol were fabricated according to a pharmaceutical recipe, and in vitro release tests showed a cumulative release of 92–100% of pirfenidone after 3 h in both common shake flask setups and bespoke apparatuses (introduced by Tietz [ 79 ]). 3D-printed ovules (alginate-based) released 82–90% of pirfenidone in a shake flask and 70–75% in bespoke apparatus. Ex vivo mucoadhesive and disintegration tests also revealed that ovules containing Macrogol and Witepsol completely dissolved within 3 hours. 3D-printed ovules disintegrated partially and fully in 3 and 8 h, respectively, which suggests longer retention time in comparison with standard excipients. It was hypothesized that pirfenidone might be absorbed by the mucosa and into the systemic circulation, but further investigation and studies to validate this assumption seem necessary. Stress urinary incontinence (SUI), a non-threatening but bothersome and quality-of-life-decreasing phenomenon, occurs due to the weakening of the sphincter, urethral muscles, and pelvic floor [ 80 ]. A vaginal pessary is considered a promising treatment to solve this issue instead of surgery. In addition, pelvic organ prolapse (POP) occurs when one or more organs in the pelvis slip, such as the bladder, uterus, bowel, and top of the vagina, and lose their normal position [ 81 ]. Altogether, SUI and POP are among the most prevalent dysfunctions that affect one-third of women in the world [ 82 ]. The most common treatment solution is considered surgery, which includes the transvaginal replacement of mesh implants to reinforce weakened urethras and prolapsed organs for SUI and POP, respectively [ 80 ]. Therefore, finding an alternative material with ideal properties and ease of fabrication appears to be crucial. Thermoplastic polyurethane (TPU) is proposed to be a satisfactory alternative to conventional polypropylene (PP) because of its chemical stability, non-biodegradability, and desired printability [ 83 – 85 ]. Eder et al. [ 86 ], using an HME method, fabricated filaments from pharmaceutical-grade TPU that were incorporated with acyclovir (as a hydrophilic model active pharmaceutical ingredient) for the preparation of vaginal pessaries by an FDM printer. Among the 3 types of TPU grades (TPU-60D, TPU-94A, and TPU-42D-35), TPU-94A showed a promising candidate to print customized vaginal pessaries in terms of mechanical properties, acyclovir release kinetics, and hydrophilicity. In a related study, researchers prepared TPU70 filaments with 0, 0.25, and 1% of 17-estradiol (E2) using a similar HME method [ 87 ]. An FDM printer then used the filaments to print meshes with different layers and geometries (Fig. 6 –A, B, and C). As shown in Fig. 6 –D, the linear release profile of E2, regardless of drug loading percentage, was achieved for 15 days, which indicated the outstanding potential of FDM-based devices for the preparation of personalized implants. Similarly, TPU filaments containing levofloxacin (LFX) were also prepared by Domínguez-Robles [ 88 ]. The printed meshes significantly decreased the bacteriostatic activity against both S. aureus and E. coli cultures, thereby minimizing the risk of post-implantation infections. Silicon-based materials were also used to print pessaries for POP-suffering females, and clinical results (done separately in Canada and Taiwan) demonstrated that using 3D printing technology to prepare patient-specific vaginal pessaries improves patient satisfaction and quality of life [ 89 , 90 ]. Fig. 6 ( A ) CAD model of a two-layer circles and lines mesh, ( B ) Two-layer circles and squares mesh, ( C ) One-layer circles and squares mesh. ( D ) Cumulative E2 release profile from tpu [ 87 ] (permission was granted to reuse the figure from Elsevier) ( A ) CAD model of a two-layer circles and lines mesh, ( B ) Two-layer circles and squares mesh, ( C ) One-layer circles and squares mesh. ( D ) Cumulative E2 release profile from tpu [ 87 ] (permission was granted to reuse the figure from Elsevier) 3D printing technology can also be applied in the fabrication of sufficient single- or multi-purpose implants for hormone replacement therapy (HRT) [ 91 , 92 ], to prevent unintended pregnancy [ 91 , 93 ], for HIV prevention [ 93 – 95 ], and for the regulation of the menstrual cycle [ 91 ]. Fu and colleagues [ 92 ] prepared progesterone-loaded PEG-PLA/PCL [ 2 , 8 ] filaments using the HME method to print “O,” “Y,” and “M”-shaped IVRs. They reported that the “O”-shaped ring had a higher progesterone dissolution than the other geometries due to its high surface area to volume ratio, and sustained release for more than 7 days can overcome the oral administration drawback of low bioavailability. The mentioned features are crucial in HRT for menopause and hypogonadism treatment. The applicability of PLA/PCL filaments containing estrogen and progesterone was evaluated by Tappa and co-workers in the printing of pessaries in the shapes of donuts, Gellhorns, IUDs, meshes, and rods [ 91 ]. Over a week, the printed disk-shaped pessaries demonstrated extended hormonal release. Furthermore, an in vitro luciferase assay in response to estrogen on the estrogen receptor luciferase reporter T47D stable cell line was performed, and luciferase activity increased by 80.85% and 74.9% by extruded filaments and disc-shaped printed pessaries, respectively. These results demonstrated the suitable release properties, cytocompatibility, and bioactivity of filaments and printed pessaries for further applications. HIV preventive and contraceptive IVRs are another type of emerging vaginal implant for which 3D printing can play an important role in fabricating patient-specific devices with efficient release properties. For instance, Young et al. [ 93 ] fabricated the first multipurpose prevention IVR for HIV and unintended pregnancy using continuous liquid interface production (CLIP TM ). Their prepared IVRs showed the sustained release of etonogestrel, ethinyl estradiol, and islatravir (ENG, EE, and ISL) for 150 and 14 days in vitro and in vivo (sheep), respectively. The researchers found the fabricated ISL-IVRs safe for future preventive applications, using a monitoring population of peripheral CD4 T-cells and mucosal cytokine changes in macaques. As previous studies mentioned earlier, successful samples of IVR fabricated with TPU have been reported in the literature, and their tunable characteristics could be beneficial for further HIV prevention devices [ 94 , 95 ]. For instance, TPU-based reservoir-type IVRs containing hydroxychloroquine, IgG, gp120, and coumarin 6 PLGA-PEG nanoparticles (C6NP) were fabricated using a lab-developed Cartesian FDM-based 3D printer by Chen et al. [ 94 ]. By implementing a rate-controlling membrane (RCM) with tunable thickness and porous structures, along with controlling the printing perimeters, the researchers achieved a daily zero-ordered release of hydroxychloroquine ranging from approximately 23 to 261 μg/mL/day. In addition, release rates of IgG, gp120, and C6NP showed pattern and in-fill (IF) density-dependent characteristics. In vitro results demonstrated that the release rate of gp120, IgG, and C6NP almost doubled in scaffolds. Welsh and co-workers reported that TPU-based vaginal rings, fabricated with droplet deposition modeling (DDM), containing 57–62 mg of dapivirine (DPV) exhibited up to 7 times more DPV release than conventional injection-molded rings containing 190–194 mg of drug [ 95 ]. Table 3 summarizes all of the studies on using 3D printing to design gynecological drug delivery devices. Table 3 3D printed drug delivery devices for reproductive disorders treatment Ref. 3D Printing method Related disease/target tissue Ink(s) Type of study (Model) Cell components (Origin) API(s) Outcome(s) Kiseleva et al. [ 52 ] Extrusion Cervical cancer/cervix Pluronic F127/alginic acid sodium salt In vitro/ex vivo (porcine vaginal membranes) Normal human vaginal mucosa (CRL 2616), human breast cancer (BT474) cell lines and HeLa cells Therapeutic gold nanoparticles (AuNPs) The cumulative release of approximately 80% of the encapsulated AuNPs occurred in nearly 48 hours, with a maximum swelling ratio of 450% achieved in 2 hours, and the 3D-printed formulations demonstrated suitable biocompatibility on HeLa, CRL 2616, and BT-474 cells. Varan et al. [ 53 ] Inkjet printing Cervical cancer/cervix PCL In vitro L929 mouse fibroblast cells, human cervical adenocarcinoma cells Paclitaxel, cidofovir Printed film that contained both drugs showed the best anticancer behavior, due to its synergistic effect, in vitro. Varan et al. [ 55 ] Extrusion (HME, FDM) Uterine cancer/uterus PCL In vitro L929 mouse fibroblast cell line and HEC-1B human endometrial adenocarcinoma cell line Paclitaxel, carboplatin The printed device obtained sustained release of paclitaxel and carboplatin (up to 10 days). We observed a decrease in cell viability of HEC-1B human endometrial adenocarcinoma cells by over 60% after treatment with paclitaxel-carboplatin PCL filaments. Holländer et al. [ 57 ] Extrusion (HME, FFF) -/uterus PCL In vitro - Indomethacin Drug release from printed IUSs was faster than corresponding filaments. Furthermore, drug release from an IUS containing 5% drug was faster than 15, and 15% was faster than a 30% drug-loaded IUS. Genina et al. [ 56 ] Extrusion (HME, FDM) Controlled release implantable devices/uterus EVA In vitro - Indomethacin Drug release from 3D-printed SR and IUS prototypes with a 5% drug was faster than counterparts with a 15% drug. Wang et al. [ 60 ] Extrusion (DIW) Ovarian Cancer/ovary PLGA In vitro, in vivo (mice) SKOV3 and A2780 (The human Ovarian Cancer cell lines) Cisplatin CDDP-PLGA significantly increased the efficacy in terms of cytotoxicity as well as changes in apoptosis in SKOV3 and A2780 cell lines. Cho et al. [ 61 ] Extrusion Ovarian Cancer/ovary Poloxamer 407 In vitro, in vivo (mice) ES-2-luc (human ovarian cancer cells), ES-2-luc ascites (mouse ovarian cancer cells) and ES-2-luc-PTXtreated-ascites (mouse ovarian cancer cells) Paclitaxel and rapamycin The one-phase release pattern released 60% of paclitaxel and 82% of rapamycin within 24 hours. Discs containing drugs prevented post-surgical peritoneal adhesion and increased survival rate in ovarian-cancer-bearing xenograft mice. Salmoria et al. [ 63 ] SLS endometrial and ovarian cancer/ovary Polyethylene In vitro - Fluorouracil, progesterone The IUDs prepared using 5W laser power showed better flexural modulus and burst release of fluorouracil. Linear and controlled release of progesterone (~18% in 38 days) was reported. Tiboni et al. [ 65 ] Extrusion (HME, FDM) Vaginal candidiasis/vigina TPU In vitro - Clotrimazole Sustained release over a 7-day period in both 50% ethanol and vaginal fluid simulant media was observed (in compliance with the Higuchi equation of release). Moroni et al. [ 66 ] Extrusion (HME, FDM) VVC/vigina EVA In vitro - Clotrimazole, bifonazole The decrease in viability of C. albicans in the presence of clotrimazole-loaded IVR was 32 times higher than bifonazole-loaded IVR after 24 hours. Patel et al. [ 67 ] Extrusion (FDM) VVC/vigina PVA, PCL In vitro Ex vivo (goat vaginal mucosa) - Itraconazole 90% of the drug was released in 48 hours, and 80% of the insert matrix was degraded in 4 days. Utomo et al. [ 70 ] Extrusion (SSE) BV/vigina PCL- methyl vinyl ether/maleic anhydride In vitro - Metronidazole Formulations with a 60/40 and 70/30 ratio of high molecular weight (HMW) PCL to low molecular weight (LMW) PCL were able to sustain the release for 72 hours. Release in discs containing 50/50 PCL content was extended up to 9 days. Kyser, Masigol, et al. [ 72 ] Bioprinting BV/vigina Gelatin/sodium alginate In vitro VK2/E6E7 cell line Lactobacillus crispatus 10:2 (w/v) gelatin/alginate formulation with dual crosslinking by genipin and CaCl 2 was the best formulation in terms of structural stability, minimal mass loss, swelling, post-printing viability, and sustained release of microorganisms over 28 days. Arany et al. [ 73 ] Extrusion (FDM) BV/vigina TPU, chitosan/HEC, agar In vitro The human Negroid cervix epithelioid carcinoma (HeLa) cell line Jellified metronidazole or chloramphenicol Fabricated samples were biocompatible, non-toxic, and bactericidal. The dissolved API amount in metronidazole-containing samples was higher than in chloramphenicol-containing samples. Kyser, Mahmoud, et al. [ 74 ] Extrusion BV/vigina PDMS/vinyl, methyl modified silica In vitro VK2/E6E7 cell line Metronidazole Sustained metronidazole release of 4 and 27 μg/mg was obtained after 24 hours and 14 days for scaffolds cured for 4 hours at 50 °C followed by 72 hours at 4 °C. Herold et al. [ 75 ] Extrusion BV/vigina vinyl-terminated polydimethylsiloxane and vinyl, methyl-modified silica In vitro VK2/E6E7 cell line Metronidazole In vitro release results from scaffolds were best modeled by the Higuchi, Korsmeyer-Peppas, and Peppas-Sahlin models. de Carvalho Rodrigues et al. [ 77 ] Extrusion (HME, FFF) Herpes Virus Genital Infection/vigina EVA In vitro Vero cells (ATCC CCL-81-VHG) Acyclovir We observed a burst release in the first 24 hours, followed by a slow and sustained release of the drug over 80 days. In vitro biological assays showed a 99% decrease in HSV-1 replication. Teworte et al. [ 78 ] Extrusion (SSE) endometriosis and fibrotic uterine/vigina Sodium alginate/HPMC In vitro, ex vivo (Porcine vagina) Human epithelial endometriotic cells (12Z cell line) Pirfenidone The 3D-printed ovule released 82–90% of pirfenidone in the shake flask and 70–75% in the bespoke apparatus. 3D-printed ovules disintegrated partially and fully in 3 and 8 hours, respectively, which suggests longer retention time in comparison with standard excipients. Eder et al. [ 86 ] Extrusion (HME, FFF) SUI/vagina TPU In vitro - Acyclovir TPU-94A showed a promising candidate to print customized vaginal pessaries in terms of mechanical properties, acyclovir release kinetics, and hydrophilicity. Farmer et al. [ 87 ] Extrusion (HME, FDM) POP, SUI/vigina TPU, PP In vitro - 17-β-estradiol (E2) The linear release profile of E2, regardless of drug loading percentage, was achieved for 15 days. Domínguez-Robles et al. [ 88 ] Extrusion (HME, FFF) POP, SUI/vigina TPU, PP In vitro - Levofloxacin Printed meshes significantly decreased the bacteriostatic activity on both S. aureus and E. coli cultures, which minimizes the risk of post-implantation infections. Lin et al. [ 96 ] SLA POP/vigina Medical grade silicon Clinical - - 6 patients reported a significant increase in QOL with customized pessaries. Hong et al. [ 90 ] SLA POP/vigina Liquid silicone rubber Clinical - - All 8 patients reported either improvements or no change in pessary ease of use, comfort, and the feeling of support provided by the pessary. Tappa et al. [ 91 ] Extrusion (HME, FDM) HRT, contraception, regulation of the menstrual cycle/vigina PLA/PCL In vitro Human T47D/Luciferase stable cells Hormones Estrone (E1), Estradiol (E2), Estriol (E3) and Progesterone Extended hormonal release for 7 days was observed. Luciferase activity increased more with extruded filaments and disc-shaped pessaries than with other shaped pessaries. Fu et al. [ 92 ] Extrusion (HME, FDM) HRT of menopause and hypogonadism, contraceptive/vigina PEG- PLA/PCL In vitro - Progesterone O-shaped printed rings demonstrated a higher dissolution rate compared to Y-shaped and M-shaped rings. Long-term and diffusion-controlled release of progesterone for more than 7 days was observed. Young et al. [ 97 ] DLS HIV prevention and contraceptive/vigina Silicone poly(urethane) resin In vitro, in vivo(sheep) T-cell Etonogestrel, ethinyl estradiol, and islatravir Both in vitro and in vivo, we observed the sustained release of etonogestrel, ethinyl estradiol, and islatravir for 150 and 14 days. Chen et al. [ 94 ] Extrusion (HME, FDM) HIV prevention/vigina TPU In vitro Human vaginal epithelial cell line VK2/E6E7, ectocervical epithelial cell line Ect1/E6E7, and human T cell line SupT1 Hydroxychloroquine, IgG, gp120, coumarin 6 PLGA-PEG NPs A daily zero-order release of hydroxychloroquine was obtained. pattern and in-fill density-dependent release of IgG, gp120, and C6NP were obtained. Welsh et al. [ 95 ] Extrusion (HME, FDM) HIV prevention/vigina TPU In vitro - Dapivirine 3D-printed IVR (with a lower amount of loaded API) exhibited up to 7 times more API release than devices fabricated with conventional methods. API, active pharmaceutical ingredients; PCL, polycaprolactone; HME, hot melt extrusion; FDM, fused deposition modeling; FFF, fused filament fabrication; IUS, Intra Uterine System; EVA, ethylene vinyl acetate; DIW, Direct Ink Writing; PLGA, Poly (lactic-co-glycolic acid); TPU, Thermoplastic polyurethane; VVC, vulvovaginal candidiasis; EVA, ethylene-vinyl acetate; IVR, intravaginal ring; PVA, polyvinyl alcohol; SSE, semi-solid extrusion; HMW, high molecular weight; LMW, low molecular weight; BV, bacterial vaginosis; HEC, hydroxyethyl cellulose; PDMS, polydimethylsiloxane; HPMC, hydroxypropyl methylcellulose; SUI, Stress urinary incontinence; POP, Pelvic Organ Prolapse; PP, Polypropylene; SLA, Stereolithography; HRT, Hormone Replacement Therapy; PLA, Polylactic acid; PEG, Polyethylene glycol; DLS, Digital Light Synthesis; HIV, human immunodeficiency virus 3D printed drug delivery devices for reproductive disorders treatment In vitro Ex vivo (goat vaginal mucosa) API, active pharmaceutical ingredients; PCL, polycaprolactone; HME, hot melt extrusion; FDM, fused deposition modeling; FFF, fused filament fabrication; IUS, Intra Uterine System; EVA, ethylene vinyl acetate; DIW, Direct Ink Writing; PLGA, Poly (lactic-co-glycolic acid); TPU, Thermoplastic polyurethane; VVC, vulvovaginal candidiasis; EVA, ethylene-vinyl acetate; IVR, intravaginal ring; PVA, polyvinyl alcohol; SSE, semi-solid extrusion; HMW, high molecular weight; LMW, low molecular weight; BV, bacterial vaginosis; HEC, hydroxyethyl cellulose; PDMS, polydimethylsiloxane; HPMC, hydroxypropyl methylcellulose; SUI, Stress urinary incontinence; POP, Pelvic Organ Prolapse; PP, Polypropylene; SLA, Stereolithography; HRT, Hormone Replacement Therapy; PLA, Polylactic acid; PEG, Polyethylene glycol; DLS, Digital Light Synthesis; HIV, human immunodeficiency virus Recent advancements in regenerative medicine and biomedical engineering have been associated with significant progress in the development of disease models, particularly with the integration of 3D printing technology. These models provide researchers with the capacity to autonomously manipulate specific cellular and molecular variables and assess the resultant system responses [ 98 ]. In this section, we will explore the studies conducted on creating disease models of the female reproductive system using 3D printing. Furthermore, models generated using this platform exhibit exceptional efficiency when assessing drug and therapeutic responses, enabling robust statistical analysis. This, in turn, leads to reduced testing expenses and promotes the utilization of alternative physiological models as substitutes for animal testing and drug screening. Xu et al. [ 99 ] utilized two types of cells, ovarian cancer cells (OVCAR-5) and fibroblasts (MRC-5), as bio-ink materials. These cells were directly deposited onto a Matrigel TM (GFR) substrate using two distinct nozzles within a 3D printing system based on a cell biopatterning system (Fig. 7 –A). Their primary objective was to construct an in vitro model of ovarian cancer. Both groups of cells displayed a survival rate exceeding 96%. The results indicated the spontaneous formation of multicellular acini and the absence of detrimental effects caused by the bioprinting process on the cells (Fig. 7 –B). This platform also demonstrated an impressive capability to achieve controlled printing at a remarkable rate of 2 million cells per minute. Baka et al. [ 100 ] conducted the second study on creating an ovarian cancer model using 3D printing technology. Initially, they focused on optimizing a sodium alginate-gelatin hydrogel. Subsequently, they incorporated cancer cells (SKOV-3 cells) and cancer-associated fibroblasts (MeWo cells) into the hydrogel mixture, resulting in the formulation of a bioink. They then conducted a computer simulation of the tumor. Remarkably, the results showed proper cellular distribution, with cell proliferation remaining intact for up to 7 days post-printing (Fig. 7 –C and D). A high cell survival rate of 91% and robust metabolic activity post-printing indicated that the bioprinting process had no adverse effects on the cells. This bioprinting model provided an ideal environment conducive to the growth and interaction of both cancer cells and stromal cells. Fig. 7 ( A ) Schematic of an efficient ejector system consisting of a computerized stage and a pair of ejectors this setup employs two ejector heads to expel distinct cell types at the same time, specifically cancer cells (OVCAR-5) and fibroblasts (MRC-5). ( B ) The image shows acini growth after biopatterning OVCAR-5 and MRC-5 cells in bright field illumination, taken 8 days after co-culture [ 99 ]. ( C ) Histological analysis of 3D-printed structures (coculture, 1 × 10 6 cells/mL bioink) shows evolving cell distribution, forming self-assembled aggregates (black arrows). ( D ) Evolution of aggregate dimensions over time in the bioprinted structures (coculture condition) [ 100 ]. ( E ) Cell viability with manual counting on a fluorescent microscope. ( F ) Confocal fluorescence microscopy image of the live (green) and dead (red) HeLa cells observed on day 1 in the crosslinked 5% HEC-1% sa construct. Scale bar: 100 μm [ 101 ]. (permission was granted to reuse the figure from John Wiley and Elsevier) ( A ) Schematic of an efficient ejector system consisting of a computerized stage and a pair of ejectors this setup employs two ejector heads to expel distinct cell types at the same time, specifically cancer cells (OVCAR-5) and fibroblasts (MRC-5). ( B ) The image shows acini growth after biopatterning OVCAR-5 and MRC-5 cells in bright field illumination, taken 8 days after co-culture [ 99 ]. ( C ) Histological analysis of 3D-printed structures (coculture, 1 × 10 6 cells/mL bioink) shows evolving cell distribution, forming self-assembled aggregates (black arrows). ( D ) Evolution of aggregate dimensions over time in the bioprinted structures (coculture condition) [ 100 ]. ( E ) Cell viability with manual counting on a fluorescent microscope. ( F ) Confocal fluorescence microscopy image of the live (green) and dead (red) HeLa cells observed on day 1 in the crosslinked 5% HEC-1% sa construct. Scale bar: 100 μm [ 101 ]. (permission was granted to reuse the figure from John Wiley and Elsevier) The first research used 3D printing to create a cervical tumor model, combining gelatin, alginate, and fibrinogen to form a hydrogel. This hydrogel was then mixed with HeLa cells to create a bioink. The printing process was done with an extrusion-based bioprinting technique. Additionally, paclitaxel was introduced as a drug agent for the cervical cancer model. This research showed the successful creation of a cubic structure with interconnected channels using 3D printing, which proved to be conducive to the transport of nutrients, oxygen, and metabolic wastes. Cell viability of more than 94% was reported, and HeLa spheroids completely covered the hydrogel. In contrast to the two-dimensional culture, where cells exhibited a flat and elongated orientation, the incorporation of paclitaxel significantly enhanced metabolic activity, which is shown to be an advantage of the bioprinting method [ 102 ]. In the subsequent study, conducted as an extension of the previous research by the same group, a 3D-printed cervical cancer model was developed using a combination of gelatin, sodium alginate, and Matrigel. Similar to the preceding investigation, HeLa cells were integrated into the hydrogel structure before the printing process. Moreover, TGF-β was employed as the primary inducer of EMT (epithelial-mesenchymal transition) within the composition of the final tumor model. The 3D bioprinting technique was utilized in this research. Similar to the previous study, the structures maintained a cubic shape, and the formation of HeLa cell spheroids was also observed. Cell viability exceeded 97%, and following treatment with TGF-β, cell adhesion was improved, and E-cadherin protein expression was significantly decreased, thereby confirming the successful induction of the EMT process through TGF-β [ 103 ]. In a study conducted by Gospodinova et al. [ 101 ], sodium alginate and HEC (hydroxymethyl cellulose) were combined to create a printing ink. After evaluating the printing properties and rheology of the ink, they mixed it with HeLa cells to form a bio-ink. Subsequently, an extrusion-based bioprinting method was used for printing. The printed structures were crosslinked with calcium chloride, and paclitaxel, a pharmaceutical ingredient, was introduced into the mixture. The study’s findings revealed that increasing the quantity of sodium alginate in the composition led to an increase in viscosity. Due to its thixotropic properties, sodium alginate played a crucial role in the sample printing process. Cell viability displayed an inverse relationship with the sodium alginate content. The highest viability was observed in samples containing 1% sodium alginate and 5% HEC (Fig. 7 –E and F). All of the studies on the use of 3D printing for disease models are summarized in Table 4 . Table 4 Designing disease models of the reproductive system using 3D printing technologies Ref. 3D Printing Method Related Disease/Target Tissue Ink(s) Type Of Study (Model) Cell Components (Origin) API(s) Outcome(s) Xu et al. [ 99 ] Cell biopatterning system Ovarian cancer/Ovary Ovcar-5 and MRC-5 In vitro Ovcar-5 (epithelial human ovarian cancer cell line) and MRC-5 cells (normal human fibroblast cell line) - Both cell types remained viable throughout the printing process and continued proliferating after being patterned. Baka et al. [ 100 ] Extrusion Ovarian cancer/Ovary Sodium alginate/gelatin In vitro SKOV-3 cells (ovarian adenocarcinoma derived cell line) and MeWo cells (ATCC HTB-65TM, granular fibroblasts, derived from human melanoma) - Cells within the bioprinted structures sustained viability and proliferation. They self-organized into heterotypic aggregates while preserving the expression of crucial phenotype markers: PAX8 for SKOV-3 cells and FAP for MeWo cells. Zhao et al. [ 102 ] Extrusion (cell assembling system) Cervical cancer/Cervix Gelatin/alginate/fibrinogen In vitro HeLa cells Paclitaxel In 3D printing environments, HeLa cells showed higher proliferation rates, MMP protein expression, and chemoresistance. They have a tendency to form cell spheroids. The printing process results in over 90% cell survival. Pang et al. [ 103 ] Extrusion (cell assembling system) Cervical cancer/Cervix Gelatin/alginate/Matrigel In vitro HeLa cells TGF-β Cell spheroids are formed. TGF-β supplementation disrupted the cell spheroid morphology and caused it to take on a spindle shape, indicating the induction of EMT. Gospodinova et al. [ 101 ] Extrusion Cervical cancer/Cervix HEC/Sodium alginate In vitro HeLa cells Paclitaxel There was an inverse correlation between relative sodium alginate content and cell viability after printing. The disease is characterized by the excessive formation of spherical cell structures. Most cell death occurred during extrusion. API, active pharmaceutical ingredients; MMP, matrix metalloproteinase; EMT, epithelial-mesenchymal transition; HEC, hydroxyethyl cellulose Designing disease models of the reproductive system using 3D printing technologies API, active pharmaceutical ingredients; MMP, matrix metalloproteinase; EMT, epithelial-mesenchymal transition; HEC, hydroxyethyl cellulose