Three-Dimensional Bioprinting and Infertility-Related Female Reproductive System Diseases: A Review of Current and Future Applications

Abstract

Background:

Infertility has a significant impact on women, affecting them both mentally and physically. Some of the current infertility-related diseases include intrauterine adhesions, endometriosis, polycystic ovary syndrome, primary ovarian insufficiency, and cancer. While existing treatments can help slow diseases progression and improve fertility for some patients, overall recovery rates remain low. The use of three-dimensional bioprinting (3D bioprinting) is becoming increasingly popular in clinical settings due to its high precision, customizable materials, and mechanical properties. In the state of infertility, its therapeutic potential is becoming more evident.

Methods:

In this paper, we summarized the current treatment status of female infertility-related diseases, including cervical cancer, ovarian cancer, endometrial cancer, polycystic ovary syndrome, intrauterine adhesions, MRKH syndrome and other diseases, used databases such as PubMed, described the research progress and future development direction of 3D bioprinting in these diseases.

Results:

3D bioprinting technology could help repair damaged endometrial and ovarian tissue, and was able to create cell-loaded biological scaffolds to help restore the structure and function of affected organs. Furthermore, the development of organoids is opening new ways for research in regenerative medicine. It is expected that 3D bioprinting will not only be able to create organoid structures for research purposes but will also be utilized in clinical settings to effectively address infertility.

Conclusions:

3D bioprinting is gaining popularity in the clinical field due to its high resolution, adjustable composition, and mechanical qualities. Infertility-related disorders damage women by inflicting a psychological and functional double blow. According to the current research, the application of 3D bioprinting technology to help patients restore fertility function has endless possibilities in the future.

Introduction

Infertility is characterized by the failure of male or female reproductive system to produce offspring after 12 months of regular unprotected sexual intercourse [1]. According to the former study, globally, between 50 and 80 million individuals experience infertility, according to World Health Organization surveys [2]. Primary infertility rates are expected to be greater in Middle Eastern and North African countries than in Latin American countries [3]. Female infertility is a reproductive system condition primarily caused by a variety of factors, including ovulatory disorders, diminished ovarian reserve, anatomical anomalies, endocrine imbalances, genetic abnormalities, functional impairments, immunological issues, chronic illnesses, sexual conditions incompatible with coitus [2, 4, 5]. In addition to organ causes, lifestyle habits also have an impact on infertility, such as stress, depression, and other psychological factors (as shown as the Fig. 1) [1, 6].

Fig. 1.

A common cause of female infertility

Diseases related to infertility in the female reproductive system include malignant diseases and benign diseases. Malignant diseases mainly include common gynecological malignant tumors: cervical cancer (CC), ovarian cancer (OC), and endometrial cancer (EC). According to the American Cancer Society, a woman has a one in 78 chance of developing ovarian cancer over her lifetime. The majority of ovarian cancers occur in postmenopausal women, however, there is still a considerable number of instances in women of reproductive age [7]. Gynecological cancers impact various groups, especially women of childbearing age, damage their ability to conceive and have children in the future. In 2022, ovarian, uterine, and cervical cancers collectively contributed to 1,405,661 new cancer diagnoses worldwide, making up 7% of all new cancer cases. Specifically, cervical cancer accounts for 8% of new cancer cases, with 25% of newly diagnosed women being under the age of 40 [8].

Benign diseases include common gynecological diseases such as endometriosis, adenomyosis, polycystic ovary syndrome (PCOS), lynch syndrome, premature ovarian insufficiency (POI), intrauterine adhesions (IUA), etc.

Endometriosis is a complex chronic inflammatory condition that impacts around 10% of women of reproductive age globally. In Western nations, its prevalence ranges from 6% to 10%, while in some Asian countries, it may be even higher, with certain studies indicating rates of up to 15%. Endometriosis is a common cause of infertility, estimated to affect up to 10%-15% of women of childbearing age. The prevalence of endometriosis increases dramatically in infertile women, up to 25–50%, and 30–50% of patients with endometriosis suffer from infertility [9]. Endometriosis is defined by the unusual invasion of the endometrial glands and stroma into the myometrium, which results in thickening and an increase in the number of myometrial cells, ultimately causing the uterus to enlarge. Common symptoms include irregular uterine bleeding, recurrent uterine and pelvic pain, painful intercourse, and infertility [10]. PCOS is a common hormonal and metabolic condition affecting women of reproductive age, characterized by significant individual variation, with prevalence rates ranging from 6% to 15%. It is characterized by chronic anovulation and hyperandrogenism. PCOS not only causes menstrual irregularities and infertility, but also increases the risk of metabolic syndrome, type 2 diabetes, and cardiovascular disease. It is an important factor in infertility in women of childbearing age, accounting for 70–80% of infertile women without ovulation [11]. Lynch syndrome, also known as hereditary nonpolyposis cancer (HNPCC), is an autosomal dominant disorder caused by germline mutations in the mismatch repair (MMR) gene encoding essential proteins such as MSH2, MLH1, MSH6, and PMS2. In women with Lynch syndrome in the age group of 20–24 years, age-related fertility decreases after diagnosis [12]. POI is a serious disease characterized by extremely low levels of anti-mullerian hormone in the blood, depletion of ovarian reserves before the age of 40, leading to premature loss of ovarian function and early menopause. Accelerated follicular atresia often leads to low fertility or infertility associated with menstrual irregularities and high pregnancy failures [1]. IUA is a frequent reason for persistent infertility, with the primary pathological characteristics of endometrial fibrosis resulting from intrauterine procedures, infarction, and other factors. These issues can lead to irregular menstruation, amenorrhea, repeated miscarriages, and secondary infertility, significantly threatening the reproductive health of women of childbearing age [13].

Tissue engineering technology is an emerging subject that applies the principles and technologies of life science and engineering, and researches and develops biological substitutes for repairing, maintaining, and promoting the function and morphology of various tissues or organs of the human body after damage based on correctly understanding the relationship between the structure and function of mammalian tissues in normal and pathological states. As a rising technology in tissue engineering, 3D bioprinting has been studied in many diseases [14–16]. Three-dimensional (3D) bioprinting, commonly known as additive manufacturing (AM), is a revolutionary "bottom-up" manufacturing process used to produce personalized physical objects (devices) and bulk materials [17]. Charles Hull introduced stereolithography (SLA) in 1984, which marked the beginning of 3D bioprinting technology. Over time, numerous types of 3D bioprinting technologies have been invented and investigated, making bioprinting of tissues and organs a possibility [18]. These advanced technologies seek to increase the precision and effectiveness of bioprinting applications in surgical settings. The ultimate goal is to overcome current technological difficulties and create more effective tissue constructs that help more patients [19].

Based on background, we aim to introduce the development status and future prospects of 3D bioprinting in the female reproductive system, so as to provide new treatments and new hope for more infertility patients.

3D bioprinting

Bioprinting technology

Extrusion-based bioprinting is the most widely used and preferred technology for tissue bioprinting. The bioink is extruded from the printhead using mechanical pressure, pneumatic pressure, or screw drive, and stacked layer by layer according to a preset path to form a three-dimensional structure [20]. The bioink is extruded as strands or filaments rather than droplets. These strands are deposited layer by layer and merge to produce the final tissue structure. Mechanical and pneumatic systems are used to extrude the bioink through the nozzle [21].

Extrusion bioprinting has several notable advantages such as ease of handling (arguably the simplest bioprinting method); affordability (relatively cheaper and primitive bioprinters have been built with a few hundred dollars); high cell-density in printed geometries; customizability and versatility-several modified versions (e.g. sacrificial, freeform, co-axial, multi-material, continuous chaotic bioprinting) to get distinct and complex 3D structures [22]. It is often used to construct tissue engineering scaffolds, organ models, such as printing scaffold structures of skin or bones tissues. 3D bioprinting offers the possibility of creating patient-specific implants that match the geometry and mechanical properties of the respective fractured bone area by being able to create multi-layered structures that mimic the complex architecture of natural bone tissue, which can improve the stability of the implant [21].

Inkjet-based bioprinting, also known as drop-on-demand bioprinting, was the first bioprinting technology to evolve from commercial two-dimensional (2D) inkjet printers. Similar to traditional inkjet printers, bioink is sprayed to a designated position in the form of tiny droplets through hot air bubbles, piezoelectric effects, and stacked layer by layer to form a three-dimensional structure [23].

Inject bioprinting has several advantages: fast printing speed, high-throughput printing; control the size of the droplets and the location of the ejection results in high cell viability. It is often used in cell printing, drug screening model construction, such as printing cell arrays for the study of drug effects on cells [24, 25]. Zamponi M through combined inject bioprinting with hydrogels containing porcine brain extracellular matrix (BMX) to test the influence of the extracellular matrix on stem cell differentiation found that the addition of BMX preferentially influences 3D bio-printed and transplant these 3D bio-printed neural cellular structures into a mouse’s cleared mammary fat pad, where they continue to grow into larger neural outgrowths [26].

Light-Assisted 3D printing uses photosensitive bioink, under the irradiation of ultraviolet light, visible light and other light sources, through the photo-initiator to initiate a polymerization reaction, so that the ink can be quickly cured in a specific area. Light-Assisted 3D printing has high printing accuracy and can achieve accurate construction of microstructure; It can print complex three-dimensional structures with good biocompatibility [27]. It is used to fabricate tissue and organ models with fine structures, such as vascular networks, neural tissues [28, 29].

Laser-assisted bioprinting uses high-energy laser pulses to generate plasma shock waves on the surface of bioink, pushing the ink to form tiny droplets or microfilaments, which are accurately deposited to the target location to achieve the construction of three-dimensional structures. Laser-Assisted Bioprinting stands out from other bioprinting methods by employing a laser beam to generate a pressure bubble that facilitates the deposition of bioink droplets onto the printing surface [30]. The top layer of the printed bracket is transparent, usually glass, allowing for laser transmission. The middle layer is a laser-absorbing layer for metallic substances such as gold and titanium. The bottom layer is made of bioink material. The middle layer absorbs the laser beam and pushes the high-pressure droplets, exposing the ribbon to the laser beam. Laser parameters such as wavelength, intensity, pulse duration, and viscosity of the bioink determine the resolution of the printed structure [31].

Laser-assisted printing has extremely high printing accuracy, which can achieve precise manipulation at the single-cell level; There is less damage to the cells and the cell survival rate is high [31]. It has important applications in the fields of structuring highly ordered cell structures, tissue engineering, and regenerative medicine, such as the construction of cardiac tissue models with specific cell arrangements [30].

Although these printing methods have their advantages and can meet the research needs of most researchers, there are still some technical disadvantages, such as limited printing accuracy, limited material selection, and not balanced in speed and quality [21] in extrusion bioprinting; limited printing height and structure, low printing speed, limited size, high material requirements, color deviations and uneven material distribution in inject bioprinting [18]; high material cost, require post-processing after printing, small printing volume, high environmental requirements, complex operation technology, and the curing effect is not good in light-Assisted bioprinting; high cost of equipment, narrow range of materials available, low printing efficiency, long time, high laser energy, and safety risks in laser-Assisted bioprinting [32]. (as shown as Fig. 2).

Fig. 2.

3D bioprinting technology and limitations

Bioink

Bioinks are the fusion of biomaterials with cells and growth factors for bioprinting tissues. They are hydrogel-based materials that are kept in a fluid state with appropriate rheological parameters that favor bioprinting [18]. Bioinks are a key component of the bioprinting process, which determines the nature of the tissue structure of the print. Viscosity, thixotropy, pH, temperature, concentration, shear stress, surface tension, printability, stacking capacity, cell density, biocompatibility, non-immunogenicity, biodegradability, swelling rate, cell adhesion, and cell proliferation are important parameters that determine the suitability of biomaterials as links [33]. The bioink contains a crosslinker that is squeezed after activation to allow the bioink to solidify into a solid structure. There are three crosslinking mechanisms currently in use: physical, chemical, and thermal crosslinking methods. According to the curing mechanism, we classify bioinks into ionic cross-linking, temperature-sensitive, photosensitive, and shear-thinning. According to the curing mechanism, we classify bioinks into ion cross-linking [34], thermosensitive [35], photosensitive [36], and shear-thinning [37]. These materials often have a variety of biological functions, and in addition to the curing mechanism, we divide them into natural [38], synthetic [39], and composite materials [40] according to the source of the material. In addition, according to the function of bioink, it can be divided into structural bioink [41], sacrificial bioink [42], functional bioink [32], and support bioink [43] (as shown in Table 1).

Table 1.

Classification of bioinks and some representative inks

Classification	Types	Represents ink	References
Curing mechanism	Ion crosslinking	Alginate	[34]
	Thermosensitive	Gelatin	[35]
	Photosensitive	Gelatin methacryloyl (GelMA)	[36]
	Shear thins	Laponite-RD (LPN) and GelMA	[37]
Source of materials	Natural	Collagen, gelatin, hyaluronic acid (HA)	[38]
	Synthesis	Polyethylene Glycol, Polyvinyl alcohol	[39]
	Compound	Alginate and Polyethylene Glycol	[40]
Functional kinds	Structure	Collagen, alginate, decellularized matrix (dECM)	[41]
	Sacrifice	Gelatin	[42]
	Function	Cellulose derivatives	[32]
	Support	dECM	[43]

3D bioprinting usually consists of three parts: hardware, software, and material. Because of its personalization, versatility, and strong regenerative repair characteristics, it has gradually been used by more and more researchers as a research method in the pathogenesis of various diseases, agent drug delivery and other related fields. However, in the female reproductive system, the application of 3D bioprinting technology is not yet mature, therefore, we intend to summarize the role and development status of 3D bioprinting technology in regenerative medicine and infertility-related female reproductive system in recent years, to better look forward to the future development (Table 2).

Table 2.

3D-bioprinting with ovary and vagina related diseases

Related diseases	Content	Finding	References
Ovary	Using the bio3D printing technology of microporous hydrogel scaffolds, a functional biological pseudo-ovary was constructed in sterilized mice	Follicular seed scaffold had a high degree of vascularization, and the biological pseudo-ovarian retained its reproductive function after implantation in the surgically sterilized mice	[74]
Ovary	The pleural layer is coated with a thin layer of gelatin-methacrylate (GelMA). The microfluidic tubes are safely placed in the designated inlet and outlet locations. NOA63 is printed on the membrane to fill the area around the channel. Exposure to 365 nm UV light for 5 min to cure UV-sensitive polymers	The importance of the three-dimensional nature of the tumor microenvironment is emphasized as a key factor in gaining unique insights into important biological events associated with tumor growth and development	[42]
Ovary	GelMA was used as a bioink, loaded with ovarian tumor cell lines and primary cultured ovarian somatic cells for extrusion 3D printing to prepare scaffolds, mouse follicles were implanted into ovarian scaffolds, and the diameter of follicles was recorded daily. Finally, in vitro maturation was performed, and ovulating oocytes were collected for observation	Follicles can grow and ovulate successfully in a stent. GelMA material is suitable for 3D printing manufacturing. The scaffold performs well in terms of hygroscopicity, degradation kinetics, and shape fidelity	[75]
Ovary	ovarian fragments with 3D printing scaffold employing dECM-derived “bioink” containingADSCs, to restore impaired ovarian function in POI	3D-bioprinted scaffold could improve retention of ADSCs and revascularization in the grafts	[76]
Ovary	The dECM solution is mixed with a bioink solution mixed with seaweed gelatin to convert the ovarian dECM into a dECM-based bioink, and 3D scaffolds with or without POCs are printed by extrusion 3D bioprinter	the expression of germ cells in the 3D scaffold-embedded POCs group was stronger than that in the unprinted hydrogel-embedded POCs group. Illustrated: Ovarian 3D bioprinting using ovarian decm-based is a promising method for ovarian failure correction	[77]
Ovarian cancer	Cancer cells (SKOV-3) and cancer-associated fibroblasts (CAFs) were combined with 3D bioprinting to make ovarian tumor models	maintain cell viability and proliferation, enabling the rapid generation of a large number of reproducible tumor models that can be interesting tools for high-throughput drug screening applications	[78]
Ovarian cancer	A 3D microtumor model with different ratios of ovarian stromal cells and leukemia cells (HL-60) was created by 3D bioprinting using pegylated fibrinogen and alginate hydrogel bioinks	Cell ratio and culture time have a regulatory effect on tumor development, and cancer cells can be detected without significantly changing the total cell density dynamics, which is promising in cell analysis and drug screening	[82]
Ovarian cancer	Taking advantage of the material deposition and precise control of cell patterns provided by bioprinting of DLP, ovca macrophage spheroids were constructed using GelMA to mimic peritoneal spheroids, and after CPMV treatment, the bioprinted spheroids showed inhibition of OvCa progression mediated by macrophage activation	Deeps understanding of the mechanisms of intra-tumoral immunotherapy for CPMV in the context of ovarian cancer. This study also highlights the potential of high-throughput tissue models using DLP bioprinting technology to study immune therapy	[81]
Ovary cancer	A nanocomposite hydrogel biomaterial was developed to construct an ovarian cancer model by 3D bioprinting and used for drug screening	gemcitabine had a therapeutic effect on ovarian tumor cells. However, ovarian tumor models have become resistant to oxaliplatin treatment	[80]
Ovary Cancer	Implantable polymer hydrogels carrying immunostimulatory cowpea mosaic virus (CPMV) were prepared by digital light processing (DLP) printing technology. Hydrogels carrying CPMV are surgically implanted into the abdominal cavity to serve as a repository for extended-release immunotherapy for cancer	Sustained release of intraperitoneal CPMV alleviates the need for repeat dosing, and we have demonstrated efficacy in ovarian cancer in a metastatic mouse model	[79]
Vagina	Cell-free vaginal matrix (AVM) bioink for three-dimensional vaginal tissue biomimetic printing. BMSCs are encapsulated with BMSCs by mixing with 15% gelatin and 3% sodium alginate solution with AVM solution. The rats were then randomly divided into a 3D scaffold group and a 3D scaffold-embedded labeled BMSCs cell group	3D scaffold encapsulated with BMSCs had a significant effect on the vascularization and epithelialization of printed vaginal tissues, and BMSCs could obtain the phenotypes of vaginal epithelial cells and endothelioid cells. The results of this study show that the bionic three-dimensional vaginal tissue embedded in BMSCs by AVM biological chain is a promising method for vaginal reconstruction	[83]
Vagina	Preparation of 3D printed scaffolds for gelatin alginate. Bioprint alginate gelatin with different weight-to-volume ratios (w/v) to determine the formulation with the highest print resolution; The effect of different crosslinking reagents on the integrity of the scaffold was also evaluated. proliferation of live bacteria over 28 days, without impacting viability of vaginal epithelial cells	provides in vitro evidence for bio-3D printed scaffolds as a novel strategy to maintain probiotic delivery, with the ultimate goal of restoring vaginal lactobacilli after microbial disturbances	[84]
Vagina	a unique bioink composed of porcine vaginal extracellular matrix (vECM), GelMA, and silk fibroin (SF) was developed to facilitate the bioprinting of vaginal stents. This vECM-GelMA-SF bioink efficiently replicates the microenvironment in vivo, supporting the in vitro culture of 3D-bioprinted vaginal stents	the use of bio-3D printed vaginal stents can significantly improve the function of the reconstructed vagina, promoting angiogenesis, rapid epithelialization, muscle regeneration, glycogen secretion, and nerve repair. The reconstructed vaginal tissue of the three-dimensional cell-loaded scaffold set is similar to that of natural vaginal tissue	[85]

Current treatments for infertility disorders

Treatment of cancer

Surgery

Endometrial cancer (EC) is among the most prevalent cancers in women, and its occurrence has been rising in recent years. The typical approach for treating patients with early EC involves a total hysterectomy and bilateral Salpingo-oophorectomy, which may or may not include lymphadenectomy. Although the 5-year survival rate is quite high, this procedure results in a permanent loss of fertility for patients. With trends like delayed marriage and childbearing, along with the existing two-child policy, an increasing number of young women are seeking ways to maintain their reproductive capabilities [44].

The possibilities for fertility preservation in OC patients are extremely restricted. OC is more frequently seen in women who have gone through menopause, but it can also affect younger women who are still menstruating. Traditional treatments can be invasive, and the likelihood of cancer returning may not justify this method [7]. Fertility-sparing surgery has gained acceptance over time, and various alternative surgical techniques have shown success in treating cancer while preserving fertility and maintaining low recurrence rates [45]. Among the different fertility preservation methods available for cancer patients, oocyte and embryo cryopreservation are the only suitable options for those with ovarian cancer.

For CC, hysterectomy is still considered the standard surgical approach [8]. As a result, women diagnosed with CC during their reproductive years frequently experience infertility as a consequence of the cancer surgery. Fertility-sparing surgery (FSS) is a feasible choice for young women diagnosed with early-stage cervical cancer, achieving a fertility preservation success rate of over 90% [46]. FSS typically contains performing staged procedures to secure at least the opposite ovary and uterus, with the goal of preserving ovarian tissue and the uterus [47]. Furthermore, FSS can enhance patients' sexual function and mental well-being, as well as support the maintain of their reproductive capabilities.

Chemotherapy or radiotherapy

Chemotherapy medications are effective in destroying cancer cells, but they can also harm healthy cells, particularly the follicular cells in the ovaries. Numerous chemotherapy agents trigger apoptosis in these follicular cells, leading to a reduction in follicular reserve. For instance, cyclophosphamide, a frequently used chemotherapy drug, can significantly reduce the count of primordial follicles in the ovaries. As the damage to the follicles progresses, ovarian function will slowly deteriorate, leading to decreased estrogen production, menstrual irregularities, and potentially amenorrhea, which can result in infertility [48].

Radiotherapy is a method that employs high-energy rays to eliminate cancer cells. When the reproductive organs are within the area being treated, they can suffer direct harm from the radiation. For instance, administering radiation to the pelvic region exposes the ovaries and uterus to these rays. The ovaries are particularly fragile to radiation therapy, even low doses can result in ovarian failure. Additionally, exposure of the uterus to a specific amount of radiation can cause changes like thinning of the endometrium and fibrosis of the myometrium, which can diminish the uterus's ability to accept an embryo and hinder embryo development [49].

Targeted therapy and immunotherapy

Targeted therapy is a method of treatment that targets a specific target of cancer cells. Although targeted therapy drugs are relatively precise and cause less damage to normal tissues, some drugs may still have some effects on fertility. For example, some targeted anti-angiogenic drugs may affect the blood supply to the ovaries and ovarian function [50]. However, there are still relatively few studies on the effects of targeted therapy drugs on fertility, and the specific mechanism and degree of influence need to be further clarified.

Immunotherapy is a treatment that fights cancers by activating the autoimmune system. The effect of immunotherapy on female fertility is unclear. On one hand, immunotherapy may not directly damage the reproductive organs as much as chemotherapy and radiotherapy [51], On the other hand, activation of the immune system may trigger some autoimmune reactions that may affect the reproductive system, but this is rare [52]. With the widespread use of immunotherapy, more research is needed to assess its long-term effects on fertility (Fig. 3).

Fig. 3.

Current treatments for infertility-related disorders

Stem cell therapy

Stem cell therapy is a developing treatment that has demonstrated various functions in cancers. Mesenchymal stem cells possess the ability to migrate directionally towards cancers tissues, making them suitable for transporting therapeutic agents, such as chemotherapy drugs and gene therapy vectors, directly to the cancer site. This targeted approach can increase the local concentration of the drug within the cancer, enhance its effectiveness against cancer cells, minimize its distribution to other areas of the body, and reduce the associated toxic side effects [53]. Researchers used mesenchymal stem cells carrying chemotherapy drugs to treat cervical cancer and found that they can accumulate at the cancer site and release drugs, effectively inhibit cancer growth, and cause less damage to normal tissues [54]. In the study of ovarian cancer, it has been found that mesenchymal stem cells can regulate the cytokine network in the tumor microenvironment, reduce the secretion of immunosuppressive factors, and increase the expression of anti-tumor immune factors, thereby enhancing the body's anti-tumor immune response [55].

Stem cells can self-renew and differentiate in multiple directions, can differentiate into a variety of tissue cells, and participate in the repair and regeneration of damaged tissues. Mesenchymal stem cells can differentiate into intestinal epithelial cells, repair chemotherapy-induced intestinal mucosal damage, and improve digestion and absorption in patients [56]. At the same time, hematopoietic stem cells can promote the recovery of bone marrow hematopoietic function, increase the number of peripheral blood cells, and alleviate bone marrow suppression after chemotherapy [57].

Although stem cell therapy has shown some potential in the treatment of gynecological malignant tumors, it is still in the research and exploration stage, and there are still many problems to be solved, such as the source, preparation process, in-depth study of the mechanism of action, and long-term safety of stem cells.

Pharmacotherapy

Pharmacotherapy in malignant tumors is mainly reflected in the application of chemotherapy and targeted and immunological drugs, including preoperative neoadjuvant chemotherapy, advanced recurrences, and metastasis. Patients with EC also use progestogen therapy to preserve fertility [58].

Observational therapy

For cancer patients, observational treatment is not passive. For example, if CC is found during pregnancy, if it is early and the condition is relatively stable, it can be intervened after the fetus matures under close observation and treatment monitoring, to take into account the safety of the mother and baby as much as possible [59]. For patients with advanced ovarian cancer, observation is also a strategy when the disease is relatively stable and enters the palliative care phase after multiple lines of therapy [60], at this time, the main focus is on the patient's symptoms and quality of life, and through regular evaluation, according to the changes in the condition, decide whether further treatment is needed, and avoid adverse effects caused by overtreatment.

Treatment of benign diseases

Surgery

Surgical treatment plays an important role in a variety of gynecological benign diseases, which can effectively eliminate lesions, alleviate symptoms, and improve the quality of life of patients [61]. However, whether to use surgery and which surgical method to choose requires doctors to formulate a personalized treatment plan according to the patient's age, condition, fertility needs, physical condition, etc.

Stem cell therapy

Stem cells can home to ovarian tissue, differentiate into ovarian cells, secrete a variety of growth factors and cytokines, promote the development and maturation of follicles in the ovary, improve ovarian function, increase the secretion level of estrogen and other sex hormones, and alleviate symptoms [62]. For diseases such as thin endometrium and intrauterine adhesions, stem cells can differentiate into endometrial cells, promote endometrial repair and regeneration, increase endometrial thickness, improve female fertility, improve menstrual cycle and menstrual flow, etc. [63].

Although stem cell therapy has shown certain potential in the treatment of gynecological diseases, most of the current research is still in the stage of basic experiments and clinical trials, and there are still problems such as immunogenicity, tumorigenicity, and unclear mechanism of action, which need further in-depth research and exploration to ensure its safety and efficacy, and promote its wide application in clinical practice.

Pharmacotherapy

For benign diseases, most patients still take drug therapy as the first choice, but most of these drugs are hormonal drugs, and taking them can have adverse effects for long time, such as low estrogen symptoms, including hot flashes, night sweats, bone loss, etc. At the same time, it may also affect ovulation function, leading to menstrual cycle disorders, making it difficult for women to forecast the time of ovulation, thus indirectly affecting the chance of conception. Commonly used drugs include gonadotropin-releasing hormone analogs (GnRH-a) [64], oral contraceptives [65], progestogens [66], etc.

Observational therapy

For physiologic ovarian cysts that are small in diameter (typically less than 5 cm), observational treatment is usually recommended. Most physiologic cysts disappear spontaneously within 1–3 menstrual cycles. Regular repeat ultrasounds can identify the nature and changes of the cyst and avoid unnecessary surgery. For smaller, asymptomatic fibroids, observational treatment is the mainstay of management. Because most uterine fibroids grow slowly, especially in patients who are nearing menopause, the fibroids may shrink gradually as estrogen levels decline [67]. Through regular gynecological ultrasound examinations, the size, location, and number of fibroids are observed, and if the fibroids do not enlarge significantly or show symptoms, special treatment can be temporarily waived.

Observational therapy is not a passive waiting, but an active monitoring and management strategy, which requires doctors to make a comprehensive assessment according to the patient's specific condition, age, fertility needs, physical condition, and other factors, formulate a personalized observation plan, and fully communicate with the patient to ensure that the patient understands and cooperates with the various examinations and follow-up during the observation process.

Infertility-related conditions, regardless of the treatment provided, can only enhance important hormone levels and alleviate clinical discomfort, but they do not achieve the main goal for patients seeking to have biological children. There is still a need for new treatments for those experiencing infertility.

3D bioprinting and infertility-related disease

3D bioprinting and uterine-related diseases

Infertility diseases associated with the uterus usually include uterine fibroids, intrauterine adhesions, endometriosis, cervical cancer, etc. In 2019, Paul K and his colleagues through 3D bioprinting combined endometrial mesenchymal stem cells (eMSCs) within hydrogels and molten electro-spun meshes to develop tissue-engineered structures. They discovered that the molten electrospinning (MES) mesh produced at 100 °C and a speed of 20 mm/s had the largest open aperture (47.2 ± 11.4 μm) and the thinnest chain thickness (121.4 ± 46 μm), which promoted optimal attachment of eMSCs. The tensile properties of the optimized MES (100 °C) was further investigated showed a Young’s modulus of 5.29 ± 0.12 MPa and ultimate tensile strength of 163.72 kPa. This indicated that the MES mesh is stiff enough to maintain the bioprinting eMSCs and that the implantation structure is structurally stable after subcutaneous implantation. The aloe vera-sodium alginate (AV-ALG) composite hydrogel was optimized to a 1:1 ratio (1% AV-1% ALG), and eMSCs obtained from human endometrial biopsies were printed onto the MES-printed mesh using this hydrogel. An assessment of the acute in vivo foreign body response in NSG mice indicated that eMSCs printed on the MES construct improved tissue integration, enhanced eMSC retention, and displayed an anti-inflammatory M2 macrophage phenotype, as evidenced by the colocalization of F4/80⁺ and CD206⁺ markers. In addition, the presence of eMSCs further reduced the response of M1-type inflammatory macrophages compared to the MES-Hydrogel mesh (P = 0.0455), but at the same time increased the overall cell flow into the mesh [68]. Their study indicated that focusing on 3D bioprinting eMSC-MES nets could effectively tackle the problems associated with non-degradable knitted fabrics in current POP treatments.

In 2020, researchers used 3D bioprinting produced a porous hydrogel scaffold infused with human-induced pluripotent stem cell-derived mesenchymal stem cells (hiMSCs). When this scaffold was implanted into a model simulating endometrial injury, it initiated an early immune response in the host. Furthermore, the implantation of the 3D-bioprinted hiMSC-loaded scaffold not only aided in the recovery of the endometrial tissue structure and the restoration of both endometrial and endothelial cells, but also improved markers of endometrial receptivity. This process partially reinstated the functions necessary for embryo implantation and the maintenance of pregnancy in the injured endometrium [69]. Their research indicated that hydrogel scaffolds loaded with 3D-bioprinted hiMSC-loaded scaffold could serve as a potential material for endometrial repair, opening up new avenues for investigation in IUA studies. In 2022, Wen et al. [70] employed 3D-bioprinted hydrogel and sustained-release microsphere (SRM) system created a 3D-bioprinted G-CSF-SRM system (3D microsphere) in vitro. In Sprague–Dawley rat IUA model, they found that these 3D microspheres enhanced local endometrial regeneration, especially, reduced endometrial tissue fibrosis, and promoted the growth of endometrial cells (both epithelial and stromal) and blood vessels. All of these researches showed that 3D-dimensional microspheres can significantly enhance endometrial receptivity and restore the reproductive function of the damaged endometrium.

Nie et al. [71] used extrusion 3D bioprinting technology created a double endometrial construct made of sodium alginate and hyaluronic acid hydrogel (Alg-HA) for endometrial regeneration. The upper layer of 3D bioprinting endometrial construct was a monolayer of endometrial epithelial cells (EECs), while the lower layer was a reticular structure containing endometrial stromal cells (ESCs). In a partial full-thickness hysterectomy rat model, after double endometrial construct transplanted, they through H&E staining to assess the morphological and structure recovery of endometrial tissue, including the thickness of the endometrium and the number of glands, and muscle fibers. It was found that at 30 days after surgery, the endometrium in the 3D-bioprinted group (235.69 ± 5.20 μm) was thicker than that in the injury (123.60 ± 3.99 μm, P < 0.001) and non-printed groups (88.20 ± 21.80 μm, P < 0.001). The endometrial thickness in rats receiving 3D bio-printed was 274.10 ± 41.50 μm, which showed a significant difference from the sham group (P < 0.001) 90 days after surgery. In addition, on day 90 post-transplantation, the number of glands per cross-section in the 3D bio-printed group (20.5 ± 4.7) was significantly higher than that in the injury group (11.0 ± 2.9, P < 0.05), and higher than that in the non-printed group (15.0 ± 2.0, P > 0.05). The quantification of muscle fibers in the 3D-bioprinted group (18.09% ± 0.41%) was significantly higher than that in the injury (13.01% ± 0.66%, P < 0.001) and non-printed groups (14.07% ± 0.76%, P < 0.001). Angiogenesis also measured. The result showed that the density of blood vessels in the 3D bio-printed group (27.78 ± 5.54) was higher than that in the non-printed (21.33 ± 2.40, P > 0.05) and injury groups (14.17 ± 4.95, P < 0.05) at the injured sites. About in vivo functional validation, they found that pregnancy rates were 93.75% (15/16) in the 3D-bioprinted group and 87.5% (7/8) in the injury group compared to 100% (10/10) in the sham group. These results indicated that 3D bioprinting technology not only restored the morphology and structure of the uterine wall but also greatly enhanced reproductive results in the surgical area following implantation. In structure, biological function, vascularization, and in vivo functional validation, three studies showed the same trend [68–70].

In recent research, 3D spheroids were prepared by extrusion 3D bioprinting and photocuring post-processing using agarose gel matrix and alginate matrix, and the spheroids successfully proliferated and self-organized into complex structures, forming a sustainable system that mimicked the condition of the tissue through the accumulation of extracellular matrix [72]. With the further research, 3D bioprinting not only focuses on the treatment of diseases but also gradually pays attention to the structural construction and functional recovery of the uterus. The most recent research developed a layered tissue engineering scaffold that replicated the structure and function of uterine tissue. This was achieved by creating multilayer biomimetic scaffolds using solvent casting, layer-by-layer assembly, and 3D bioprinting techniques. The scaffold's deformed layer was made from a communion of poly-lactate-co-trimethyl carbonate (PLATMC) and polylactic acid-co-glycolic acid (PLGA), to simulate the myometrium's function in uterine tissue. After etching the surface of the PLATMC/PLGA, a multilayer film of estradiol (E2), polydopamine (PDA), and hyaluronic acid (HA) was formed on the scaffold to create an intelligent drug delivery system for controlled and sustained E2 release. A layer of hydrogel, consisting of gelatin methacryloyl (GelMA) and gelatin (Gel) and containing bone marrow mesenchymal stem cells (BMSCs), was 3D-bioprinted onto the PDA@E2/HA-20 multilayer membrane, completing the scaffold's construction. In a 37 °C aqueous environment, the scaffold transitioned from a flat to a tubular structure, fulfilling the requirements for curved stents in uterine tissue engineering. The elongations at break of PDA@E2/HA-10 and PDA@E2/HA-20 scaffolds were 498.20 ± 17.42% and 501.37 ± 22.98%. With the formation of PDA@E2/HA multilayer films on scaffolds, the tensile strength of PDA@E2/HA-10 and PDA@E2/HA-20 scaffolds increased to 1.49 ± 0.08 MPa and 1.62 ± 0.12 MPa, respectively. There is no large difference for this property among the scaffolds. This illustrates that the PDA@E2/HA multilayer films on scaffolds improved the strength and modulus of scaffolds but did not impair their elasticity. This biomimetic scaffold featured a layered structure, curved shape, high stretchability, and controllable continuous E2 release, indicating significant potential for applications in uterine tissue regeneration [73] (as summary as Fig. 4).

Fig. 4.

3D bioprinting and uterine-related infertility research

While 3D bioprinting has significantly progressed in capturing the complexity of tissues, it's crucial to recognize that the optimization process can be lengthy and challenging. Creating successful models requires careful consideration of various experimental factors, including the selection of suitable printing techniques and biomaterials, cell growth, the biological environment, microarchitecture, and the functionality of the tissue.

3D bioprinting and ovarian-related diseases

Ovarian-related infertility diseases include ovarian cancer, ovarian cysts, polycystic ovary syndrome POI, etc. In recent years, there has been an increasing number of studies on 3D bioprinting and ovarian-related diseases, providing new treatments for the treatment of various diseases.

In 2017, a functional biological pseudo-ovary was built in sterilized mice using 3D bioprinting technology of microporous hydrogel scaffolds, with the follicular seed scaffold having a high degree of vascularization, and the biological pseudo-ovarian retained its reproductive function after implantation in the surgically sterilized mice. Mechanical properties were necessary to create biomaterial implants that are easy to handle during surgery and that closely match native ovarian tissue stiffness (mean compressive elastic modulus was 16.84 kPa) [74]. Microfluidic components and spheroids were created through microfluidics, 3D bioprinting, and photocuring post-processing methods, making them suitable for drug screening or assessing the cytotoxicity of biochemical substances. The biomanufacturing methods employed in study was ideal for quickly prototyping in vitro tumor models that were relevant to physiological conditions, and there was potential for fully automating the entire process [42]. In 2022, GelMA was used as a bioink, loaded with ovarian tumor cell lines (COV434, KGN, ID8) and primary cultured ovarian somatic cells for extrusion 3D printing to prepare scaffolds, mouse follicles were implanted into ovarian scaffolds, and the diameter of follicles was recorded daily. Finally, in vitro maturation was performed, and ovulating oocytes were collected for observation [75], they found that follicles could grow and ovulate successfully in a scaffold. GelMA material was suitable for 3D bioprinting manufacturing. The scaffold performs well in terms of hygroscopicity, degradation kinetics, and shape fidelity.

In 2021, We mixed the decellularized matrix(dECM) solution within bioink, which solution mixed with gelatin and sodium alginate, then printed 3D scaffolds with or without primary ovarian cells (POCs) by extrusion 3D bioprinting. It was found that the expression of germ cells in the 3D scaffold-embedded POCs group was stronger than that in the unprinted hydrogel-embedded POCs group. It’s illustrated that ovarian dECM-based bioink was a promising material for ovarian failure correction through 3D bioprinting [76]. Furthermore, hormone evaluation showed that the serum E2 level in the 3D-bioprinted scaffold encapsulating POCs group (465.91 ± 24.77 pg/ml) was significantly higher than that in the ovariectomized group (332.28 ± 26.17 pg/ml, P = 0.000) and the 3D scaffold group (390.06 ± 41.47 pg/ml, P = 0.014), which was close to that in the non- ovariectomized group (494.31 ± 35.96 pg/ml, P = 0.292) and the hydrogel-encapsulated POCs group (424.69 ± 24.26 pg/ml, P = 0.138). After implantation of the 3D-bioprinted scaffold encapsulating POCs, FSH (44.69 ± 24.17 mIU/ml) and P (2.55 ± 1.34 ng/ml) returned to physiological levels (FSH: 50.34 ± 2.73 mIU/ml; P:3.35 ± 2.56 ng/ml). Serum P levels in the 3D-bioprinted scaffold group (12.75 ± 2.59 ng/ml) and hydrogel-encapsulated POCs group (14.52 ± 3.02 ng/ml) were similar to those in ovariectomized mice (12.75 ± 2.59 ng/ml). Then, our team combined drug-free in vitro activation (IVA) and 3D-bioprinted adipose-derived stem cells (ADSCs) to create a scaffold to restore the ovarian function of rats with POI [77],we detected higher CD31-positive signals and functioning blood vessels blood vessels in the 3D-bioprinted engineering ovary group, as compared with the other groups. Additionally, a larger number of positively stained cells for CD31 was found at 1 week after transplantation and also increased significantly at 4 weeks. The findings indicated that 3D-bioprinted scaffolds could increase ADSCs retention and graft revascularization. In addition, we constructed a 3D-bioprinted engineering ovary consisting of drug-free IVA and ADSCs for the first time, contributing to a more successful strategy for restoring ovarian function in POI rats [77]. Due to the specificity of ovarian structure and function, there is currently a lack of evidence in vivo functional verification (pregnancy outcome).

Ovarian cancer models were created by combining cancer cells (SKOV-3) with cancer-associated fibroblasts (CAFs) using 3D bioprinting technology. These tumor models had shown the capability to sustain cell viability and growth, allowing for the quick production of numerous reproducible tumor models that could serve as valuable resources for high-throughput drug screening [78]. Implantable polymer hydrogels containing the immunostimulatory cowpea mosaic virus (CPMV) were created using digital light processing (DLP) printing technology. Regarding the stiffness of the hydrogels, mechanical tests confirmed that the blank group and the CPMV-laden hydrogels exhibited comparable Young's modulus (sham gels:6531 ± 628.0 Pa, CPMV gel: 6117 ± 2670 Pa, P = 0.8065). This suggests that CPMV was incorporated into the hydrogel by a simple mixture, which results in a diffusion-based release mechanism. The hydrogels were surgically inserted into the abdominal cavity to act as a reservoir for prolonged immunotherapy for cancer. The continuous released of CPMV into the peritoneal cavity, which reduced the necessity for multiple doses, had shown effectiveness in treating ovarian cancer in a transferred mouse model [79]. A nanocomposite hydrogel biomaterial was developed to construct an ovarian cancer model by 3D bioprinting and used for drug screening. The nanocomposite bioink consists of aldehyde-modified cellulose nanocrystals (ACNC), aldehyde-modified hyaluronic acid (HA), and gelatin. By controlling the ratio of ACNC to gelatin, the hydrogel has adjustable gelation time, mechanical properties, and printability. In addition, hydrogel-embedded ovarian cancer cells have a high survival rate and a fast growth rate. The results showed that gemcitabine had a therapeutic effect on ovarian tumor cells. However, ovarian tumor models have become resistant to oxaliplatin treatment [80]. Using the precise control of material deposition and cell patterning provided by DLP-based bioprinting, used GelMA to mimic peritoneal spheroids, and after CPMV treatment. The 3D-bioprinted spheroids showed inhibition of OC progression mediated by macrophage activation. The research showed that CPMV could regulate and activate macrophages, induces OC cell death and restore normal cell–cell junctions [81]. For deep understanding of the mechanisms of intra-tumoral immunotherapy for CPMV in the context of OC. A study also highlights the potential of high-throughput tissue models used DLP bioprinting technology to study immunotherapy. A 3D microtumor model was developed using 3D bioprinting with pegylated fibrinogen and alginate hydrogel bioinks, incorporating varying proportions of ovarian stromal cells and leukemia cells (HL-60) [82]. This study found that tumor growth was influenced by both cell ratio and culture period. Furthermore, cancer cells could be recognized without significantly changing the overall cell density dynamics, providing opportunities for cell research and medication testing. Because current research on malignancies is currently focused on drug delivery, there is a lack of stability studies.

3D bioprinting and vagina-related diseases

There was a limited amount of research on vaginal diseases associated with infertility, with the majority of studies concentrating on vaginal reconstruction. In 2021, our team first used cell-free vaginal matrix (AVM) bioink for 3D-bioprinted vaginal tissue. Bone marrow mesenchymal stem cells (BMSCs) were encapsulated with 15% gelatin and 3% sodium alginate solution with AVM solution. The rats were then randomly divided into a 3D scaffold group and a 3D scaffold-embedded labeled BMSCs group. We found that the 3D scaffold encapsulated with BMSCs had a significant effect on the vascularization and epithelialization of 3D-bioprinted vaginal tissues, and BMSCs could obtain the phenotypes of vaginal epithelial and endothelioid cells. The results of this study showed that the bionic 3D-bioprinted vaginal tissue embedded in BMSCs by AVM biological chain was a promising method for vaginal reconstruction [83].

Recent years, the study of intestinal flora has been hot, and it has also shown good advantages in vaginal reconstruction. Kyser AJ and colleagues developed 3D-bioprinted scaffolds using gelatin and alginate. They experimented with various weight-to-volume ratios (w/v) of alginate and gelatin to identify the formulation that achieved the best print resolution. Additionally, they assessed how different crosslinking agents influenced the structural integrity of the scaffolds. The 3D-bioprinted scaffolds that included L.Crispate demonstrated a sustained release and growth of live bacteria over 28 days while maintaining the viability of vaginal epithelial cells. This study provided evidence for 3D-bioprinted scaffolds as a novel strategy to maintain probiotic delivery, with the final goal of restoring vaginal lactobacilli after microbial disturbances [84].

In our latest study, a unique bioink, which composed of porcine vaginal extracellular matrix (vECM), GelMA, and silk fibroin (SF), was developed to facilitate the bioprinting of vaginal stents. This vECM-GelMA-SF bioink efficiently replicated the microenvironment in vivo, supporting the in vitro culture of 3D-bioprinted vaginal stents. In situ studies showed that the use of 3D-bioprinted vaginal stents could significantly improve the function of the reconstructed vagina, promote angiogenesis, rapid epithelialization, muscle regeneration, glycogen secretion, and nerve repair. The reconstructed vaginal tissue of the 3D cell-loaded scaffold set was similar to that of natural vaginal tissue [85].

Limitation

Although 3D bioprinting has shown good therapeutic prospects, there are still many problems that need to be solved. Limitations can be divided into 3D bioprinting and clinical applications.

3D bioprinting

Technical

Printing accuracy: It is difficult to completely and accurately replicate the microstructure and complex interactions between cells in the human reproductive organs, which may lead to certain deviations in the morphology and function of the printed tissues or cells, affecting their normal physiological functions [86].

Cell survival and maturation: Although printed cells can survive in the short term, there are still many challenges in order to keep them alive and mature in vitro for a long time, forming fully functional germ cells or tissues and organs [87, 88].

Vascularization challenges: vascularization is a key issue when constructing large 3D-bioprinted reproductive organs. Without an effective vascular network, it is difficult for printed tissues to obtain sufficient nutrients and oxygen, and metabolic waste products cannot be discharged in time, which will limit the growth and function of tissues [89].

Material

Biomaterials safety: Biomaterials used for 3D bioprinting should be harmless to human body and have no side effects such as immunogenicity or toxicity [90]. However, the safety of some materials in the long-term use process still needs to be further verified, and there may be potential risks.

Material performance matching: it is not easy to find a printed material that fully matches the biomechanical properties and biocompatibility of human reproductive tissue [91, 92]. The mismatch of material properties may affect the structural stability and function of the printed tissue.

Ethics and legal level

Ethical controversy: 3D bioprinting of germ cells or organs involves many ethical issues, such as reproductive rights, gene editing, gender selection, and so on.

Legal gaps and regulatory problems: The legal norms and regulatory system of related technologies are not perfect, and there is a lack of clear legal provisions on the application scope, operational norms and intellectual property rights of 3D bioprinting in infertility treatment, which brings some difficulties to the clinical application and promotion of technology.

Clinical application

Safety assessment

3D bioprinting products require comprehensive safety assessments, including short-term and long-term safety observations, before use in clinical treatment. However, due to the novelty and complexity of the technology, there is a lack of perfect evaluation standards and methods, and it is difficult to accurately judge the safety and stability of the 3D-bioprinted tissue or organ in the body, and whether it will cause other potential health problems [93].

Validation of treatment effect

To demonstrate the effectiveness of 3D bioprinting in the treatment of female infertility-related diseases requires extensive clinical trials and long-term follow-up observations. However, there are relatively few relevant clinical studies and the sample size is limited, making it difficult to fully verify its therapeutic effect and long-term prognosis, which also limits the clinical popularization of this technology to some extent.

Cost and accessibility

The research development and application cost of 3D bioprinting technology is high, including equipment purchase, material research and development, and training of professional and technical personnel, which makes related treatment expensive and difficult to popularize. Only a few patients can benefit, thus affecting the wide application of technology and social fairness.

Future applications

Future research directions of 3D bioprinting in diseases

The research prospect of 3D bioprinting in female infertility-related diseases is very broad. In uterine-related diseases, 3D bioprinting could create highly biomimetic endometrial tissue for repairing damaged endometrium [71]. By printing biomaterials containing endometrial stem cells, extracellular matrix, and growth factors, the regeneration and repair of the endometrium can be promoted, the receptivity of the endometrium can be improved, and good conditions for embryo implantation can be created, to solve the infertility caused by thin endometrium and intrauterine adhesions [68, 94, 95]. It can also establish a personalized endometrial model for each patient, which can be used to study the physiological and pathological state of the patient's endometrium, predict the possibility of embryo implantation, and formulate a personalized treatment plan. At the same time, these models can also be used for drug screening to find more effective drugs for the treatment of endometrium-related diseases. At the same time, with the help of 3D-bioprinted endometrial tissue, we can further study the occurrence and development mechanism of endometrial diseases, such as endometriosis, endometritis, etc. By simulating the endometrial microenvironment in disease states, the pathophysiological process of diseases can be revealed, which provides a theoretical basis for the development of targeted treatments [96].

In ovarian-related diseases, 3D bioprinting technology was used to accurately construct ovarian tissues that are highly matched with the patient's physiological characteristics. By obtaining patients' cells, such as ovarian stem cells or other related cells, with suitable biomaterials, ovarian tissues with normal physiological functions can be printed for the treatment of early-onset ovarian insufficiency and other diseases, providing the possibility for patients to restore ovulation and endocrine function [76, 77]. In addition, for women who require chemoradiotherapy for diseases such as cancer, ovarian tissue can be constructed and preserved in vitro using 3D bioprinting technology before treatment [97]. Once the patients finish their treatment, the 3D-bioprinted ovarian tissue is reintroduced into the body to help maintain the patient's fertility and prevent the ovarian function damage that can result from chemotherapy and radiotherapy, which can lead to infertility. Currently, research on organoids is advancing rapidly, and there is hope that 3D bioprinting will soon be utilized to produce functional ovarian organoids. Future optimizations are anticipated to enhance their resemblance to the functions of actual ovaries [98]. For example, the whole process of follicle development, maturation, and ovulation can be better simulated in printed ovarian organoids [99], providing a more ideal in vitro model for studying the pathogenesis of ovarian diseases, drug screening, and developing new treatments.

In vagina-related diseases, our team not only made vECM but also made vECM-GelMA-SF bioink for printing vaginal stents. Studies have shown that the bioink could effectively replicate the in vivo microenvironment, promote stem cell viability and enhance neovascularization, and in animal models, the use of 3D-bioprinted vaginal stents can significantly improve the function of reconstructed vagina, promoting angiogenesis, rapid epithelialization, muscle regeneration, glycogen secretion, and nerve repair [85]. In terms of drug delivery, researchers at the University of Nottingham in the United Kingdom had developed 3D-bioprinted pirfenidone-containing semi-solid vaginal ovules for the treatment of endometriosis. This ovule has the characteristics of slow and controlled drug release and good mucosal attachment in vitro and in vivo, which can reduce the metabolic activity of endometriosis epithelial cell lines in vitro [100]. In addition, 3D bioprinting technology could be used to build a diagnostic model of vaginal diseases, providing doctors with an intuitive and visual reference, and can be personalized according to the specific situation of patients, effectively simulating the pathological characteristics of vaginal diseases, and improving the accuracy and timeliness of diagnosis in the future. With the development of technology, 3D bioprinting will be the greatest benefit to patients if they can more accurately customize personalized treatment plans according to each patient's vaginal anatomy, physiological characteristics, and disease conditions in the future, such as customizing reconstruction stents that are more suitable for the patient's vaginal morphology, targeted drug delivery carriers, etc., to improve the treatment effect and patient comfort.

Future research directions on the limitations of 3D bioprinting

Due to some limitations of 3D bioprinting technology, in order to solve the challenges in the treatment of female infertility-related diseases, measures can be taken from technological innovation, material research and development, ethical and legal norms, and clinical application optimization. In terms of technological innovation, improvements can be considered in the future in terms of improving printing accuracy, overcoming vascularization problems, and optimizing cell culture technology. In the field of material research, generating new biomaterials and reducing their immunogenicity are critical steps toward improving biocompatibility. To meet ethical and legal requirements, it is critical to improve ethical evaluations and legal oversight to permit faster clinical applications in the future. For clinical applications, promoting early use of 3D bioprinting technology can be accomplished by establishing safety evaluation criteria, conducting multi-center clinical trials, lowering costs, and increasing accessibility, with the ultimate goal of better addressing the fertility issues faced by infertile patients.

Conclusion

In conclusion, 3D bioprinting is gaining popularity in the clinical field due to its high resolution, adjustable composition, and mechanical qualities. Infertility-related disorders traumatize women by inflicting a psychological and functional double blow. According to the current research, the application of 3D bioprinting technology to help patients restore fertility function has endless possibilities in the future.

Authors’ contributions

YL: contributed to design, draft preparation, writing, data curation, tables and figures; YM: contributed to investigation; LW and CL contributed to methodology; XH: contributed to supervision; JZ: proof read the manuscript.

Data availability

The data that supports the findings in this study are available from the corresponding authors upon reasonable request.

Declarations

Conflict of interest

The authors declare they have no competing interests.

Ethical approval

There are no animal experiments carried out for this article.