Research on the mechanism of human umbilical cord mesenchymal stem cells and their extracellular vesicles in the treatment of common reproductive diseases

Abstract

Reproductive system disorders significantly contribute to infertility, and traditional or conventional treatments often have limited efficacy in addressing this issue. In recent years, stem cell therapy has emerged as an alternative therapeutic strategy owing to its various advantages. Human umbilical cord mesenchymal stem cells (hUC-MSCs) are pivotal in tissue repair owing to their robust proliferative capacity, potent immunomodulatory effects, low immunogenicity, and paracrine actions. Extracellular vesicles (EVs), the primary mediators of paracrine functions, exhibit therapeutic effects similar to those of hUC-MSCs. Consequently, numerous researchers have investigated the application of hUC-MSCs and their EVs in treating reproductive disorders. These cells have the potential to restore fertility by mitigating oxidative stress, excessive autophagy, and ferroptosis in tissues, while promoting the expression of anti-inflammatory factors and vascular remodeling. However, hUC-MSCs present significant limitations compared to EVs, including higher tumorigenicity and low infusion efficiency. Consequently, EVs may emerge as the primary alternative therapy, while hUC-MSCs hold promise as a therapeutic option with potential applications in regenerative medicine.

Introduction

In recent years, reproductive system disorders have affected the health and well-being of millions of couples worldwide [1]. In the female reproductive system, the ovaries and uterus play a crucial role in both natural conception and in vitro fertilization [2]. Healthy ovaries are essential for ensuring oocyte quality and maturation in females. Once the oocytes merge with sperm to form zygotes, they develop in the fallopian tubes before being implanted into the uterus; consequently, any phenotypic abnormalities or functional disorders in these structures can lead to infertility [3, 4].

Stem cells and extracellular vesicles (EVs) are currently utilized in various areas of disease treatment. For instance, stem cells can address acute myocardial infarction by releasing biological factors, whereas mesenchymal stem cell (MSCs)-derived EVs can significantly inhibit tumor cell growth [5–7]. Moreover, enhancing the effectiveness of stem cells in disease treatment has become an important area of interest. For example, the use of hematopoietic stem cells that express tumor-associated antigen-specific T-cell receptors or chimeric antigen receptors is being explored for treating acute myeloid leukemia [8].

Stem cells are a population of cells with self-renewal and multilineage differentiation potential that can be differentiated into at least one type of highly differentiated daughter cell in a controlled manner [9]. These cells are typically categorized as embryonic stem cells, adult stem cells, and induced pluripotent stem cells. However, the ethical controversies associated with embryonic stem cell research and clinical applications, coupled with the inefficiency and teratogenic potential of induced pluripotent stem cells, present significant challenges [10, 11]. Mesoderm-derived MSCs have been widely used in the treatment of various diseases because of their strong proliferation ability, pluripotency, genomic stability in vitro, and lack of ethical issues [11, 12]. (As shown in Fig. 1)

Fig. 1.

Stem cell differentiation process. The inner cell mass obtained from the blastocyst differentiates into the primitive ectoderm (with significant expression of Nanog, OCT4, and SOX2). Following a series of divisions, it further differentiates into the endoderm, ectoderm, and mesoderm. At this stage, the expression of OCT4 and Nanog decreases, while mesoderm-specific markers T-protein and Mix Paired-Like Homeobox (MIXL1) increase. The mesoderm then undergoes further division to yield MSCs and hematopoietic stem cells.

(source of photo: Figdraw)

Among the various sources of MSC, the vascular matrices of bone marrow and adipose tissue have been widely studied and used. Since then, umbilical cords and placenta, which belong to adult tissues but have younger biological characteristics, have gradually become important sources for obtaining MSCs. There are significant differences in the accessibility, content, proliferation rate, immunomodulatory capacity, and cytokine secretion profiles of these MSC sources. (1) Bone marrow mesenchymal stem cells (BM-MSCs) were the first MSCs to be identified and isolated, although they are challenging to obtain from the body. BM-MSCs demonstrate excellent expansion ability and multidirectional differentiation potential in vitro, making them a longstanding focus of scientific research and clinical applications [13]. (2) Compared with BM-Mscs, adipose-derived mesenchymal stem cells (AD-MSCs) are more readily accessible, involve minimal tissue damage, have a straightforward isolation process, and exhibit significantly higher proliferation efficiency. These characteristics make AD-MSCs a promising option for stem cell therapy [14]. (3) Human umbilical cord mesenchymal stem cells (hUC-MSCs) were successfully isolated from human umbilical cords by Han Zhongzhao in 2006. These cells exhibited a higher proliferative capacity than BM-MSCs, while maintaining a similar cytokine profile. As umbilical cords are considered medical waste, hUC-MSCs not only alleviate the discomfort associated with tissue harvesting but also circumvent ethical constraints, thereby establishing them as a significant focus in the field of cell therapy [15]. The advantages and disadvantages of the three types of cells are compared in the following aspects: (1) Expression markers, Mebarki [16] et al. evaluated the immunophenotypic characteristics of cell cultures at passage 3 and found that more than 90% of the cells expressed CD10, CD29, CD44, CD73, CD90, CD105, and HLA-ABC; CD146 was only expressed by hUC-MSCs, CD34 was only expressed by AD-MSC, and CD133 was more expressed in BM-MSC and AD-MSC. High expression of CD146 enables cells to have a strong attachment ability and may be more advantageous in specific therapeutic environments. (2) Secretome composition: hUC-MSCs secrete higher levels of hepatocyte growth factor (HGF), basic fibroblast growth factor-2 (FGF-2), and nerve growth factor (NGF). HGF stimulates the proliferation of various cell types, induces the formation of tubular structures to facilitate angiogenesis, exhibits anti-apoptotic and anti-fibrotic properties, and promotes cell migration. FGF-2 synergizes with vascular endothelial growth factor (VEGF) to stimulate endothelial cell proliferation and migration, contributing to the formation of new vascular, tissue repair, and maintenance of stem cell pluripotency. Nerve growth factor (NGF) plays a significant role in the treatment of nerve injuries [17, 18]. (3) Proliferation and senescence: hUC-MSCs have a higher proliferation rate and rapid population doubling time than BM-MSC and AD-MSC. hUC-MSCs can replicate rapidly during in vitro culture and significantly increase the number of cells in the short term [18]. hUC-MSCs exhibit very low expression levels of HLA class I molecules in inflammatory environments. When stimulated by IFN-γ, hUC-MSCs did not express HLA-DR compared to BM-MSC, indicating that hUC-MSCs have lower allogeneic immunogenicity in inflammatory diseases and are more suitable for clinical treatment [18, 19].

The therapeutic efficacy of MSCs in disease treatment is primarily attributed to their paracrine activity rather than their differentiation into specific cell types. This suggests that MSC-EVs may significantly contribute to the therapeutic effects of MSCs, as they serve as crucial mediators of intercellular communication and reservoirs for growth factors, immunomodulators, and other bioactive molecules [20]. Generally, the biological functions of EVs are analogous to those of MSCs. EVs are a class of particles secreted by cells, characterized by a lipid bilayer, and are incapable of self-replicating. They have been identified as transient carriers of various molecules between cells, a property that holds significant potential for advancing fundamental biological research and developing biomarkers and therapeutic applications [21]. To enhance global research on EVs, the International Society for Extracellular Vesicles was established in 2011 and produced three guidance documents published in 2014, 2018, and 2023, respectively. The latest version not only improves the 2018 perspective but also adds suggestions and guidance for new development areas in the field. It also extensively covers the nomenclature, pretreatment variables, isolation and characterization of EVs, and in vitro and in vivo analysis of EVs release, uptake, and function. 2023 For EVs nomenclatures and related terms, the 2023 version proposes many, while this review mainly focuses on EVs and their subtype classification [22]. The generation process of EV subtypes is different and independent, mainly covering three categories: exosomes, microvesicles, and apoptotic bodies. Exosomes originate from the invagination of the cell membrane, mature into late endosomes, and bud inward to form vesicles. They assemble into multivesicular bodies that fuse with the cell membrane to release secretions known as exosomes. In contrast, microvesicles bud directly from the cell membrane and are approximately 100–2000 nm in size. Apoptotic bodies, which are approximately 1–5 μm in size, are produced by the breakdown of apoptotic cells and often occur during programmed cell death [23]. Therefore, EVs can be isolated and concentrated based on their biophysical properties, such as size, density, charge, and surface composition. Common isolation techniques include: (1) size exclusion chromatography, which separates many non-vesicular extracellular particles (NVEPs) and EVs based on their size. (2) Differential ultracentrifugation is used to segregate EV subtypes of varying sizes or densities according to specific centrifugation parameters. (3) Commercial kits are specifically designed for EVs and can isolate mixtures of Eps. Following isolation, it is essential to characterize EVs to assess their quantity and purity, as well as to evaluate the impact of non-EV components on the EV preparation [22]. Currently, they are assessed using widely accepted physical properties and protein marker methods, which include identifying EV size, concentration, and morphology using high-resolution microscopy, light scattering, charge, and flow cytometry methods. As EVs are secreted or released from cells via budding or endosomal transformation, they retain a portion of the original cell membrane and cytoplasm of the parent cell. This facilitates the assessment of protein markers to determine whether the target transmembrane protein originates from cellular polyvesicular bodies or endosomal organelles and whether the proteins transferred from the cytoplasm to EV are consistent. This method can also be used to measure common protein contaminants that may be separated from EVs, such as apolipoproteins, albumin, and uroregulin. Common methods include immunostaining, imaging-based light scattering, and imaging-based flow cytometry. However, to date, no protein has been identified that can be used to characterize all types of EVs, nor has any protein been found to be consistently present in every EV within a given EV subtype, which is a major limitation [22, 24].

Multiple studies have applied hUC-MSCs and their exosomes to repair bone, skin, and peripheral nerve injuries, revealing their ability to effectively improve post-injury organ function in the brain, kidneys, and liver [25–27]. Consequently, studies have explored the connection between hUC-MSCs and reproductive organ injuries, delving into the therapeutic mechanisms of hUC-MSCs and offering a novel curative approach for intractable conditions that traditional medicine struggles to resolve [28]. By exploring the mechanisms underlying hUC-MSC and EV therapies for common reproductive system disorders, we can gain deeper insights into the advantages of stem cell therapy. This may contribute to reducing infertility rates, increasing the probability of live births, and providing a theoretical foundation for expanding hUC-MSC applications to treat other related diseases. Therefore, this review summarizes the potential mechanisms of hUC-MSCs in treating ovarian, uterine, and testicular tissue disorders and methods to enhance their therapeutic efficacy in these conditions (As show in Fig. 2).

Fig. 2.

Types and modalities of hUCMSCs/ hUCMSC-Evs for the treatment of reproductive diseases. EVs obtained from hUC-MSCs after umbilical cord isolation and culture, along with their therapeutic effects on spermatogenesis abnormalities caused by testicular injury and torsion, including antioxidant stress, anti-apoptotic, anti-inflammatory, and prevention of excessive autophagy; therapeutic effects of hUC-MSCs and EVs on intrauterine adhesions (IUA) via anti-inflammatory action, fibrosis reduction, and endometrial reconstruction, alongside the IUA progression process: endometrial injury, abnormal repair and fibrosis, adhesion formation; therapeutic effects of hUC-MSCs and EVs on premature ovarian failure and polycystic ovary syndrome (PCOS) through anti-inflammatory action, prevention of excessive autophagy, immune modulation, fibrosis inhibition, and metabolic regulation.

(source of photo: Figdraw)

Treatment pathways for hUC-MSCs

hUC-MSCs

Research has shown that hUC-MSCs exert therapeutic effects through three primary mechanisms. Inflamed sites release cytokines and chemokines that stimulate hUC-MSC migration, EVs, and cytokine secretion. These components exert paracrine effects on adjacent cells and play key roles in angiogenesis, cell proliferation, anti-inflammation, immunomodulation, anti-apoptosis and anti-fibrosis. These processes are crucial for the recovery and regeneration of damaged tissues [29].

Numerous studies have demonstrated that in conditions of ischemia, hypoxia, or injury, both endogenous and exogenous MSCs exhibit a propensity to localize preferentially to the site of injury, which is a critical strategy [30, 31]. It has been observed that MSCs adhere to endothelial cells in a manner dependent on P-selectin. However, unlike leukocytes, MSCs do not express P-selectin ligands; instead, they utilize glycoproteins and galectin-1. Due to the relatively low affinity of P-selectin on the MSC surface for specific oligosaccharide chains of glycolipids or glycoproteins and the potential influence of blood flow velocity, MSCs exhibit rolling motion within blood vessels. Cellular activation during MSC homing is typically facilitated by a G protein-coupled chemokine receptor that interacts with wound-secreted cytokines to initiate integrin adhesiveness and mediate MSC migration. Stromal cell-derived factor-1 (SDF-1, also known as CXCL12) is a small chemokine within the CXC chemokine family that plays a crucial role in MSC transport and homing to the injury site. Upon injury to blood vessels or tissues, SDF-1 levels increase significantly and bind to the C-X-C motif chemokine receptors 4 (CXCR4) and CXCR7 expressed by MSCs. This interaction induces MSC mobilization along the SDF-1 concentration gradient, facilitating their homing to damaged tissues and exerting therapeutic effects [32, 33].

The adhesion of MSCs to the endothelium is associated with the activation of integrins, which occurs during the interaction between chemokines and their receptors. MSCs express a wide range of integrins, including β1, β2, α1, α2, α3, α4, α5, α6, and αV. The noncovalent assembly of the α4 and β1 subunits is essential for the formation of very late antigen-4 (VLA-4), which plays a crucial role in MSC adhesion to inflamed endothelial cells and can be activated by SDF-1. For instance, research has shown that increasing the activity of the α4 subunit in the wrong place helps transplanted MSCs locate and attach to bone injury sites in mice. Given that the β1 subunit is abundantly expressed by MSCs, the subsequent assembly of these two subunits leads to an increased expression of functional VLA-4 on the cells, for which vascular cell adhesion molecule-1 serves as a ligand. This interaction suggests that these two components facilitate MSC rolling and firm adhesion [34]. Following firm adhesion to the endothelium, MSCs migrate along the inner surface of the blood vessels in response to a chemotactic gradient. This process requires exogenous factors, including fluid shear stress and chemokines at the target site. Shear stress derived from flow has been shown to enhance the stasis of both endothelial cells and MSCs, potentially mediated by increased integrin activation. Chamberlain et al. [35]. observed a significant increase in the percentage of crawling MSCs in the presence of flow and chemokines. Ultimately, to complete endothelial migration, a cell must traverse three barriers: endothelial cells, the basement membrane, and the pericellular cavity. During this process, MSCs must penetrate the endothelial cell layer by secreting matrix metalloproteinases (MMPs), with MMP2, MMP9, and MT1-MMP playing crucial roles [36].

hUC-MSCs at the site of injury secrete cytokines, chemotactic molecules, growth factors, and extracellular matrix components to regulate inflammation and apoptosis, initiate progenitor cell proliferation, and stimulate tissue repair, thereby influencing target cells and creating favorable conditions for cell survival [37]. Cytokines and exosomes are the primary components of stem cell paracrine signaling [38]. In most tissues, restoration of blood flow at the lesion site is crucial for regenerative efficiency to occur. One mechanism by which MSCs ameliorate various diseases is by mediating angiogenesis through the secretion of biologically active factors that interact with membrane receptors on adjacent cells [39]. hUC-MSCs are enriched with a range of pro-proliferative and anti-apoptotic factors, including Transforming Growth Factor-β (TGF-β1), Epidermal Growth Factor (EGF), granulocyte and granulocyte-macrophage colony-stimulating factors, platelet-derived growth factor, VEGF and associated chemokines [40, 41]. According to this study, the VEGF family can directly stimulate the proliferation and migration of endothelial cell populations, which is a significant factor in revascularization [42]. Additionally, another crucial mechanism of hUC-MSCs is their anti-fibrotic capability. HGF is the primary bioactive factor identified in the secretome of hUC-MSCs and is believed to exert anti-apoptotic and pro-mitotic effects across various tissues. Its co-secretion with TGF-βs may be pivotal to the anti-fibrotic properties of the MSCs population [43, 44].

hUC-MSCs exhibit low immunogenicity owing to their minimal expression of HLA-DR and MHC class I molecules [45]. Furthermore, hUC-MSCs lack the co-stimulatory molecules CD80 and CD86, which are essential for T-cell activation and survival, indicating that hUC-MSCs do not provoke acute rejection and are suitable for cell-based allogeneic therapy [46]. Although the precise mechanisms of action of hUC-MSCs remain to be fully elucidated, both direct cell-to-cell interactions and the secretion of soluble factors play crucial roles in their immunomodulatory functions [47]. Notably, hUC-MSCs exert significant immunosuppressive effects, particularly on effector T cells, by secreting potent immunosuppressive factors, such as prostaglandin E2 (PGE2), indoleamine 2,3-dioxygenase (IDO), and TGF-β1. IDO can be induced by the pro-inflammatory cytokine IFN-γ, catalyzing the conversion of tryptophan to kynurenine, which effectively inhibits T cell proliferation [48, 49]. hUC-MSCs can inhibit the activation of M1-type macrophages through the secretion of IL-6 and HGF, which in turn suppresses dendritic cell differentiation and maturation. Additionally, they induce IL-10 production by monocytes and promote the generation of M2-type macrophages via TNF α-mediated activation of cyclooxygenase-2 and TNF-stimulated gene-6 [50–52]. M2-type macrophages are crucial for mitigating inflammatory responses, facilitating tissue repair, and maintaining immune homeostasis [53]. Although the immunosuppressive effects of hUC-MSCs have been documented in numerous studies, the underlying mechanisms are predominantly derived from in vitro data, indicating that further investigation is warranted to fully elucidate these mechanisms. (As shown in Fig. 3)

Fig. 3.

Therapeutic pathways of hUC-MSCs/ hUC-MSC-EVs. Following the substantial release of SDF-1 at the site of injury, intravenously administered hUC-MSCs and EVs adhere to CXCR7 and CXCR4 receptors expressed on the MSC surface. Upon reaching the site through processes of rolling, activation, arrest, crawling, and migration, MSCs traverse the endothelial cell layer by secreting MMP9, MMP2, and MT1-MMP, ultimately arriving at the injury site. At this location, MSCs facilitate therapeutic effects through paracrine mechanisms, primarily enhancing tissue repair and regeneration via immune modulation and cytokine secretion.

(source of photo: Figdraw)

hUC-MSC-EVs

A growing body of evidence suggests that hUC-MSCs primarily exert therapeutic effects through paracrine mechanisms. Similarly, EVs derived from hUC-MSCs have been shown to exhibit comparable therapeutic potential. EVs can deliver functional molecules, including microRNAs, long non-coding RNAs, and proteins, to recipient cells. Through intricate intercellular communication, these EVs facilitate angiogenesis, mitigate fibrosis, and promote tissue remodeling [54]. This functionality may be attributed to the membrane structure of EVs, which is enriched with receptors or ligands that can interact with target cells, thereby endowing them with intrinsic targeting capabilities. However, most therapeutic EVs are rapidly cleared by the mononuclear phagocyte system, such as macrophages, in vivo, resulting in off-target distribution and reduced therapeutic efficacy. Enhancing the targeting specificity and selectivity of EVs remains a significant challenge. To address the limitations associated with the insufficient expression of natural targeting ligands, various active targeting strategies have been developed [55]. (1) Ligand/peptide display technology involves the genetic engineering of exogenous targeting peptides or proteins to fuse with EV membrane proteins (e.g., Lamp-2b, CD9, CD63, CD81, or LA). This modification enables EVs to efficiently display specific ligands, thereby significantly enhancing their recognition and binding to target cells. (2) Aptamers offer advantages over antibodies and peptides, such as high stability, strong specificity, low toxicity, and excellent tissue penetration ability. They can bind to targets with high affinity, demonstrating their broad potential for use in targeted therapies. For instance, an E3 aptamer that specifically recognizes prostate cancer cells has been successfully conjugated to siRNA-loaded EVs, effectively inhibiting cancer cell proliferation and metastasis in both in vitro and in vivo experiments. (3) Antibody-mediated targeting strategies significantly enhance recognition accuracy and therapeutic specificity by displaying antibody fragments (e.g., scFv and nanobodies) or full-length antibodies on the surface of EVs via chemical conjugation or membrane fusion [56, 57]. Currently, engineered EV therapeutic strategies are primarily applied in cancer and central nervous system disease treatments; however, they offer a promising avenue for the management of reproductive disease.

hUC-MSCs and EV for the treatment of androgenital disorders

Spermatogenesis is a multifaceted cellular developmental process in which spermatogonia undergo a sequence of successive mitotic divisions, meiosis, and morphological differentiation within the seminiferous tubules of the testes, ultimately resulting in the formation of mature spermatozoa. This process is regulated by the coordinated action of numerous signaling molecules, and disruption by any harmful factors, including genetic, endocrine, environmental, and drug-related influences, can impair male fertility [58, 59]. Conventional treatments for male infertility primarily focus on improving the sperm quality. For instance, individuals with idiopathic male infertility may undergo treatment with anti-androgens and gonadotropin therapies. During the preconception period, antioxidants, such as vitamin E and zinc, are commonly administered to boost conception rates. With technological advancements, assisted reproductive technologies have been increasingly utilized to treat infertility, complementing traditional methods that enhance intrauterine fertilization rates [60]. However, these methods are ineffective in cases of infertility caused by gamete deficiencies. Therefore, the emergence of stem cell therapy offers new hope. For instance, Hermann et al. [61] discovered that transplanting spermatogonial stem cells into the testes of chemotherapy-treated macaques increased the number of functional sperm in the testes. Additionally, hUC-MSCs exhibit antioxidant properties, suggesting that they may possess the potential to restore male fertility.

hUC-MSC therapy

Rano et al. [62] suggest that embryonic stem cells bear a strong resemblance to MSCs found in the testes. These cells reside in the basal layer of the seminiferous tubules within the testes and are capable of asymmetric division to produce progenitor cells, which under certain conditions, trigger cellular differentiation. This suggests that transplanted MSCs may interact with these cells to restore fertility. Fatemeh et al. [63] found that in vitro co-culture of hUC-MSCs with mouse testicular cells (mTCs) resulted in significantly reduced expression of POU Class 5 Homeobox 1 (Oct4), whereas DEAD-Box Helicase 4 (vasa, Ddx4), Fragilis, and sycp3 were markedly elevated. Oct4 is highly expressed in embryonic stem cells, whereas Vasa, Fragilis, and Sycp are germ cell markers. This indicates that hUC-MSCs have the potential to differentiate into germ cells in vitro. Yang et al. [64] directly injected hUC-MSCs into the testicular stroma of azoospermic mice. This intervention successfully reversed the abnormal expression of meiosis-stage-specific genes and testicular germ cell marker genes, including Deleted In Azoospermia Like (Dazl), Ddx4, Stimulated By Retinoic Acid Gene 8 (Stra8), Cyclin A1, Transition Protein 2, Phosphoglycerate Kinase 2, Testis expressed gene 18, A-Kinase Anchoring Protein 3, Vasa, synaptonemal complex protein (Scp3), Piwi-like protein 1 (Miwi), among others. This further demonstrates that hUC-MSCs exert their therapeutic effects in vivo primarily through paracrine mechanisms rather than direct differentiation into spermatogonia. The following sections explore the impact of UC-MSCs on infertility by examining their effects on immunoregulation, antioxidative stress, anti-apoptosis, and autophagy inhibition.

Liang et al. [65] transplanted hUC-MSCs into a testicular torsion injury model and found that MSC therapy not only significantly reduced the expression of inflammatory mediators such as Tumor Necrosis Factor (TNF-α) and IL-1β in spermatogenic cells and the degree of neutrophil infiltration, but also suppressed torsion-induced oxidative stress and germ cell apoptosis, thereby significantly improving the testicular Johnson score (an effective method for assessing spermatogenic function). To elucidate the paracrine mechanism of hUC-MSCs, researchers co-cultured their conditioned medium (hUC-MSC-CM) with human umbilical vein endothelial cells (HUVECs). The results showed that in TNF-α-stimulated HUVECs, hUC-MSC-CM significantly downregulated the mRNA expression levels of TNF-α, IL-1β, and E-selectin. This effect likely stems from the active cytokine-rich components of hUC-MSC-CM. This experiment, similar to the findings of Yang et al. [64], indicates that hUC-MSCs primarily exert immunomodulatory and antioxidant effects in vivo via paracrine mechanisms.

Zhang et al. [66] restored paclitaxel-induced sperm damage in mice using hUC-MSCs. They found that hUC-MSCs not only improved the expression of proliferation-related proteins and meiosis-associated proteins in mouse testes, including Proliferating Cell Nuclear Antigen (PCNA), Synaptonemal Complex Protein 3 (SYCP3), MutL Homolog 1 (MLH1), DNA Meiotic Recombinase 1 (DMC1), and REC8 Meiotic Recombination Protein, while also increasing the expression of antioxidant proteins such as Superoxide Dismutase 1 (SOD1), Catalase (CAT), Peroxiredoxin (PRDX), and BCL-2 Apoptosis Regulator (BCL-2) in the testes. It also reduced the expression of BCL-2 Associated X, Apoptosis Regulator (BAX), a protein that affects germ cell apoptosis, thereby restoring blastocyst formation and normal embryonic development. This demonstrates that hUC-MSCs can act on the injury site through the homing effect, participating in the regulation of the testicular antioxidant microenvironment via the Sirtuin 1/ Nuclear factor erythroid 2-related factor 2(NRF2)-SOD1/CAT/PRDX pathway. Huang et al. [67] found that hUC-MSCs can partially reverse nitroanilide-induced reproductive damage by activating the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. This includes reducing the levels of light chain 3 (LC3), LC3II/I, BECN1 (Beclin-1) in testicular tissue and increasing the number of spermatogenic cells in testicular tissue. This study demonstrates that hUC-MSCs can improve spermatogenic abnormalities by suppressing excessive autophagy in germ cells.

hUC-MSC-EV therapy

Yue et al. [68] successfully isolated exosomes from hUC-MSC-CM using ultracentrifugation and demonstrated the high expression of surface-specific positive markers Tumor Susceptibility 101, CD9 Molecule, CD63, and CD8 via western blotting. They injected these exosomes into the testes of ICR mice treated with busulfan and found that the exosomes partially reversed busulfan-induced azoospermia by significantly upregulating germ cell-specific gene expression (e.g., vasa, miwi, Stra8, and Dazl), reducing the expression of apoptotic proteins BAX and Caspase-3, and increasing the expression of the anti-apoptotic protein BCL-2. Farzana et al. [69] reported findings consistent with those of Yue et al. [68]. Using commercial reagents, exosomes were isolated from hUC-MSC-CM and co-cultured with human testicular supporting cells (hSerCs) treated with cyclophosphamide. They discovered that exosomes significantly promoted cell proliferation, anti-apoptosis, and DNA repair. This effect was validated in mice, where chemically treated mice treated with hUC-MSC-exo exhibited restored testicular morphology and hormone levels. This mechanism likely involves the inhibition of the p38 mitogen-activated protein kinases/extracellular signal-regulated kinases/AKT (p38 MAPK/ERK/AKT) signaling pathway, reducing apoptosis and enhancing germ cell tolerance to chemotherapy.

The above studies reveal that in animal experiments, both hUC-MSCs and their exosomes can be used to treat disorders of the male reproductive system. Both treatment approaches exhibit similar mechanisms, primarily through paracrine signaling pathways. They restore testicular morphology and function by upregulating germ cell-specific gene expression, modulating immunity, counteracting oxidative stress, inhibiting apoptosis, and suppressing autophagy.

hUC-MSCs and EV for the treatment of female reproductive disorders

The ovaries are integral to the female reproductive system and are responsible for oocyte production and hormone secretion, which regulate reproductive functions. These hormones not only modulate the activities of various organs and tissues but also contribute to their maintenance and health. Ovarian function decline can result in reproductive disorders, such as reduced ovulation rates, irregular menstruation, and diminished libido. Furthermore, it may lead to abnormalities in the immune, digestive, and skeletal systems. Consequently, proactive treatment and prevention of ovarian diseases are of paramount importance [70]. Premature ovarian insufficiency (POI) and polycystic ovary syndrome (PCOS) are prevalent gynecological disorders, and individuals diagnosed with PCOS have an elevated risk of developing POI [71]. Oxidative stress-induced cellular atrophy and excessive autophagy in granulosa cells are the primary contributors to follicular dysfunction and POI. Current research suggests that MSCs can mitigate apoptosis and enhance oxidative stress [72]. Therefore, this study investigated the therapeutic effects of hUC-MSCs and EVs on damaged ovaries through mechanisms such as immunomodulation, anti-apoptosis, antioxidant stress, autophagy inhibition, and metabolic regulation.

hUC-MSCs and EVs for injured ovarian diseases

hUC-MSC therapy

Xie et al. [73] administered hUC-MSCs to a mouse model of PCOS induced by dehydroepiandrosterone (DHEA). They found that the hUC-MSC treatment group could inhibit the expression of pro-inflammatory factors, such as TNF-α, IL-1β, and Interferon-gamma (IFN-γ), increase the expression of the anti-inflammatory factor IL-10, and alleviate the pathological changes and functional impairments in the local ovarian and uterine tissues of PCOS mice. In addition, the study also revealed that hUC-MSCs could inhibit the classical M1 activation, promote the transformation of macrophages into the anti-inflammatory M2 phenotype, and significantly increase the percentage of peripheral regulatory T cells in DHEA-induced PCOS mice, while reducing the percentages of neutrophils, IFN-γ + CD19 + B cells, IFN-γ + CD4 + T cells, and IL-17 + CD4 + T cells in the spleen. It can be seen that hUC-MSCs can effectively improve the pathological changes and functions of PCOS by inhibiting local and systemic inflammatory responses in PCOS mice. Deng et al. [74], consistent with Xie’s findings [73], demonstrated that hUC-MSCs significantly downregulate the mRNA expression of pro-inflammatory factors (IL-6 and IL-1β) and apoptosis-related proteins while simultaneously upregulating the mRNA expression of anti-inflammatory factors (IL-10 and TSG-6) and angiogenesis factors (Vegf). Furthermore, in vitro experiments were conducted to explore the molecular pathways through which hUC-MSCs regulate granule cells (GCs) apoptosis. The hUC-MSC-treated group exhibited increased AKT phosphorylation and elevated p-P38/P38 ratios. As key regulators of apoptosis and proliferation, these findings suggest that hUC-MSCs may exert antiapoptotic and anti-inflammatory effects by activating the AKT and p38 pathways.

It has been discovered that reactive oxygen species (ROS) are generated during mitochondrial respiratory chain processes. Moderate ROS levels promote granulosa cell proliferation and differentiation, whereas excessive ROS levels induce apoptosis and excessive autophagy, leading to impaired follicular development. Therefore, Dai et al. [75] injected 3D spheroid-cultured hUC-MSCs into a POF rat model. They found that rats treated with hUC-MSCs not only significantly restored hormone levels, such as follicle-stimulating hormone (FSH) and estradiol (E2), but also reduced oxidative stress markers malondialdehyde (MDA) and GSH, autophagy-related proteins SQSTM1 (P62), Autophagy Related 5 (ATG5), LC3A/B, apoptosis-related proteins (BAX, BCL2), and PCNA, thereby reducing drug-induced damage to granulosa cells and restoring fertility.

Iron plays a crucial role in cellular metabolism, and iron overload can induce oxidative stress and physiological dysfunction in cells and tissues. Unlike autophagy and cell death, ferroptosis is characterized by distinct morphological and biochemical features, including mitochondrial shrinkage, cristae reduction, outer mitochondrial membrane rupture, increased intracellular free iron levels, ROS accumulation, and lipid peroxidation [76]. Research indicates that iron overload or ferroptosis not only impairs follicular development but may also induce ovarian tissue fibrosis, suggesting a close association between the two [77]. Chen et al. [78]. investigated the mechanism of hUC-MSCs in treating premature ovarian failure using in vitro experiments. They established an ovarian injury model using cisplatin and found that the chemotherapeutic agent induced excessive accumulation of ROS by depleting glutathione (GSH) and reducing glutathione peroxidase 4 (GPX4) expression. This leads to increased expression of Acyl-CoA synthetase long chain family member 4 (ACSL4), Transferrin Receptor, Nuclear Receptor Coactivator 4, α-smooth muscle actin (α-SMA), and Collagen I mRNA expression, thereby exacerbating ovarian cell apoptosis and ovarian fibrosis. Simultaneously, they treated damaged ovaries with hUC-MSCs and ferroptosis inhibitors and found that both preliminarily restored ovarian morphology and reduced fibrosis in the ovaries. This further indicates that hUC-MSCs can mitigate chemotherapy-induced ovarian damage by inhibiting ferroptosis.

Zhao et al. [79] investigated the restorative effects of hUC-MSCs on ovarian damage using in vivo experiments involving mice. In a Cyclophosphamide (CTX)-induced POI model, numerous indicators related to lipid and amino acid metabolism in ovarian tissue exhibited significant alterations, including glycerophospholipid metabolism, sphingolipid metabolism, histidine metabolism, and aminoacyl-tRNA biosynthesis. This led to a sharp decline in sphingosine synthesis, which inhibited PI3K/Akt pathway activation. PI3K, a key regulator of intracellular vesicular transport, plays a crucial role in this pathway. Its inhibition restricts lipid transport and impedes steroid hormone synthesis, disrupting glucose metabolism and resulting in a significant increase in monosaccharides. Elevated monosaccharide levels are associated with apoptosis, which exacerbates POI progression. hUC-MSC treatment activates the PI3K pathway, enhancing the transport capacity of lipid metabolites, promoting hormone synthesis and secretion, and restoring ovarian function.

hUC-MSC-EV therapy

Researchers have also investigated the effects of hUC-MSC-EVs on ovarian damage. He et al. [80]. co-cultured hUC-MSC-EVs obtained via high-speed centrifugation with human ovarian granulosa cells (KGN) treated with nitrogen mustard for 48 h. This treatment significantly suppressed apoptosis and promoted granulosa cell proliferation in the ovaries. Further quantitative proteomic analysis revealed that this effect was likely due to the high expression of clusterin in EVs. Clusterin significantly inhibited apoptosis by activating the PI3K/AKT signaling pathway.

Zhou et al. [81] isolated exosomes using high-speed centrifugation and treated cyclophosphamide-induced POI mice. These findings were consistent with those of Chen et al. [78], who found that hUC-MSC-exos restored ovarian function by increasing Nrf2 and GPX4 expression, suppressing ROS production in granulosa cells, and restoring follicle and granulosa cell numbers. Miao et al. [82] obtained hUC-MSC-EVs using a total exosome isolation kit, and administered IGF-1-carrying hUC-MSC-EVs to CTX-induced POI mouse models. Compared to the POI group, IGF-1-carrying hUC-MSC-EVs significantly mitigated CTX-induced granulosa cell damage and ovarian dysfunction in the POI group. Inhibition of IGF-1 expression reduced nuclear Nrf2 and heme oxygenase-1 (HO-1) protein levels, while it increased cytoplasmic NRF2 expression significantly. Concurrently, the levels of ROS, MDA, LC3II/I, Beclin-1, and LC3-positive cells increased, whereas the GSH content decreased. These findings indicate that hUC-MSC-EVs suppress excessive autophagy and damage in GCs via the IGF-1/Nrf2/HO-1 pathway. Zhou et al. [83]. demonstrated that hUC-MSC-EVs can mitigate excessive autophagy in ovarian cells through a different mechanism. They used ultracentrifugation to isolate EVs and co-cultured these EVs with DHEA-induced KGN. The results revealed significant reductions in LC3BII/I, Beclin-1, Parkin RBR E3 ubiquitin-protein ligase (Parkin), and PTEN-induced kinase-1 (PINK1) protein levels were significantly reduced. Methyltransferase-like 3 (METTL3), a writer enzyme for PINK1 N6-methyladenosine modification, maintains the stability of PINK1 mRNA. The PINK1/Parkin pathway is a classical route for mitochondrial autophagy. This confirms that hUC-MSC-EVs can suppress PINK1/Parkin-mediated excessive autophagy in granulosa cells by downregulating METTL3 expression. Validation in mouse models further demonstrated that hUC-MSC-EVs inhibit autophagy and enhance ovarian function through this pathway. Chen et al.‘s [84] findings align with those of Zhou et al. [81], who observed significantly elevated levels of adenosine 5‘-monophosphate (AMP)-activated protein kinase (AMPK)-phosphorylated proteins and increased LC3BII/I expression in the hUC-MSC-exo treatment group, along with markedly reduced SQSTM1 expression. Exosomes restored smooth autophagy flux, and in vitro experiments further demonstrated that AMPK inhibition led to the re-accumulation of autophagy-related proteins. This indicates that hUC-MSC-exo modulates autophagy homeostasis via the AMPK pathway.

Collectively, these studies demonstrate that hUC-MSCs and EVs primarily treat drug-induced ovarian damage through immune modulation, anti-apoptotic effects, antioxidant stress protection, and prevention of excessive autophagy. Furthermore, research by Miao [82] and Zhou [83] identified promising therapeutic targets at the clinical level, which warrants further validation and application in subsequent studies.

hUC-MSCs and EVs for injured endometrium

Successful pregnancy is contingent on the presence of normal embryos and a healthy uterine environment. Research indicates that significant endometrial damage in women of reproductive age frequently leads to intrauterine adhesions (IUA) and endometrial thinning, which may result in amenorrhea, infertility, miscarriage, and other related symptoms. Endometrial damage is frequently associated with abortion, uterine surgery, and impaired endometrial stem cell function. Consequently, aggressive treatment of such damage may enhance fertility [85]. Current therapeutic interventions, such as hysteroscopy, hormone therapy, and antibiotics, are limited in their ability to temporarily normalize the shape and volume of the uterine cavity. However, they fail to restore the structure and function of the endometrium, leading to a significant increase in the recurrence rate of severe uterine adhesions after surgery. hUC-MSCs have been extensively used to treat various tissue and organ injuries. Additionally, research has demonstrated that BM-MSCs can significantly enhance endometrial thickness through their migratory and immunomodulatory properties when applied to damaged endometrium. Consequently, hUC-MSC transplantation represents a novel therapeutic approach for addressing endometrial injury [86].

hUC-MSC therapy

Zhang et al. [87] analyzed uterine tissue treated with MSCs using a gene microarray platform. They found that after hUC-MSC transplantation, 45 miRNAs that were downregulated in the damaged endometrium were upregulated, whereas 39 miRNAs that were upregulated in the damaged endometrium were downregulated. GO and KEGG analyses indicated that endometrial injury primarily results from inflammatory responses, protein degradation, and extracellular matrix (ECM) breakdown. The restorative effects of hUC-MSCs are discussed below, focusing on immunomodulation, anti-fibrotic actions, and proangiogenic properties. Zhang et al. [88]. investigated the restorative effects of hUC-MSCs on the endometrium from the perspective of inflammatory injury. After establishing an endometrial injury model in rat uteri by injecting 95% ethanol, hUC-MSCs were administered via tail vein injection. The study revealed that hUC-MSCs promote inflammatory repair by downregulating the expression of proinflammatory factors IFN-γ, TNF-α, and IL-2, while upregulating the expression of the anti-inflammatory cytokine IL-10 and growth factors. Yang et al. [51]further investigated the anti-inflammatory effects of hUC-MSCs. They established a uterine scar model in mice by creating incisions at the uterine junction, suturing the site, and injecting hUC-MSCs into the site. The study revealed that hUC-MSCs effectively suppressed fibrosis and scar formation within the uterine scar tissue while partially restoring endometrial thickness. This effect may have arisen because, following hUC-MSC treatment, the expression of the M1 macrophage marker genes inducible nitric oxide synthase (iNOS) and TNF-α decreased in the rat uterine scar region, while the expression of the M2 macrophage marker genes Mannose Receptor C-Type 1 (CD206) and CD163 increased, accompanied by increased IL-10 infiltration. This significantly promotes macrophage polarization toward the M2 phenotype, thereby alleviating tissue inflammation. Furthermore, both studies demonstrated that hUC-MSCs promote endometrial angiogenesis and alleviate fibrosis.

Sun et al. [89]. also explored the therapeutic effect of hUC-MSCs on IUA from the perspective of the endometrium. They found that hUC-MSCs overexpressing miR-455-5p promoted the phosphorylation of Janus Kinase 2 (JAK2) and Signal Transducer and Activator of Transcription 3 (STAT3) in endometrial stromal cells (ESCs) and mouse uterine tissues by downregulating Suppressor of Cytokine Signaling 3 (SOCS3). This led to a reduction in the increase in the number of endometrial glands, inhibition of endometrial fibrosis, and alleviation of endometrial injuries. This indicates that miR-455-5p can repair endometrial injury by activating the JAK/STAT3 signaling pathway and promoting ESC proliferation and cell cycle progression.

These studies revealed that in both in vivo and in vitro models, hUC-MSCs primarily participate in alleviating uterine injury through endometrial remodeling by exerting immunomodulatory effects and miRNAs expression via a paracrine mechanism.

hUC-MSC-EV therapy

Researchers have also investigated the effects of hUC-MSC-EVs on endometrial injury, focusing primarily on their antiapoptotic, antifibrotic, and ECM remodeling properties. Wang et al. [90] isolated hUC-MSC-exos using a commercial kit to treat mifepristone-induced damage in human endometrial stromal cells (hEndoSCs). They found that stem cell-derived exosomes could be recruited to damaged tissues, where they reduced mifepristone-induced apoptosis in hEndoSCs by upregulating BCL2 and downregulating Cleaved Caspase-3 protein levels, while activating the Phosphatase and Tensin Homolog (PTEN)/AKT signaling pathway. Activation of the PTEN/AKT signaling pathway subsequently upregulates VEGF and VEGFR-2 expression, playing a significant role in the regulation of the proliferation, differentiation, and migration of endometrial cells. However, this study did not investigate the mechanism by which exosomes treat damaged hEndoSCs. Separate studies have reported that hUC-MSC-exos contain multiple miRNAs capable of inducing silencing complexes to bind the 3′-UTR of target mRNAs, thereby suppressing their expression through transcriptional repression or mRNA degradation. Consequently, studies have demonstrated that miR-7162-3p overexpression enhances the ability of hUC-MSC-exos to reduce apoptosis in damaged ESCs. This effect may arise because miR-7162-3p directly recognizes and binds to the 3′-UTR of Apolipoprotein L6 (APOL6), thereby inhibiting apoptosis [91].

Song et al. [92]also found through in vitro experiments that hUC-MSC-exos overexpressing miR-140-3p can directly target Forkhead box protein P1 (FOXP1) to regulate the TGF-β/Smad signaling pathway, thereby significantly reducing the expression levels of fibrosis-related markers in human endometrial stromal cells (HESCs), including α-SMA, Collagen Type I Alpha 1 chain (COL1A1), and Cellular Communication Network Factor 2 (CTGF). According to numerous studies, TGF-β can induce the phosphorylation of SMAD2 and SMAD3 through the activation of TGF-β receptor type I kinases, which subsequently form an oligomeric complex with SMAD4, thereby regulating the transcription of ECM-related genes to induce fibrosis. In contrast, Wang et al. [93]. found in ex vivo experiments that hUC-MSC-exos, which overexpress microRNA-202-3p, facilitate wound healing by decreasing the expression of MMP11 in the ECM in the early stage and increasing the levels of COL1A1, COL3A1, COLVI, and fibronectin to promote ECM deposition. In contrast, MMP11 is elevated at a later stage, and it is hypothesized that it may be activated by other pathways; however, the underlying mechanism of this activation remains unclear. Other studies have suggested that MMP11 remodels the local ECM environment by degrading ColVI. However, in this study, although it was the target gene of miR-202-3p, it did not seem to be significantly associated with ECM remodeling in HSCs. Thus, the paracrine therapeutic mechanism of exosomes is complex and requires further investigation.

The results of the aforementioned experiments indicate that the therapeutic effects of hUC-MSC-exos on endometrial damage are primarily mediated through miRNAs. These miRNAs regulate various pathways by targeting specific genes, thereby reducing apoptosis, decreasing endometrial fibrosis, and remodeling the ECM. This mechanism is analogous to the manner in which hUC-MSCs treat the injured endometrium and offer a safer, cell-free therapeutic strategy for numerous diseases (Table 1).

Table 1.

Mechanisms of hUCMSCs/hUCMSC-EVs in the treatment of reproductive diseases

Model	Indications	Product	Route/dose	Primary mechanism	Key outcomes	Ref.
mTCs	/	hUC-MSC	Co-culture /6 Wells	↓Oct4 ↑vasa, Fragilis, sycp3	Differentiating into germ like cells	[63]
BALB/c male mice	Azoospermia	hUC-MSC	Local testicular injection /1 × 105 cells	↑vasa, Scp3, miwi ↑Genes related to meiosis	Improves spermatogenic damage	[64]
SD male rats	Testicular torsion	hUC-MSC	Tail vein injection /1 × 105 cells	↓TNF-α, IL-1β, and neutrophil infiltration levels ↓ROS, apoptosis	Promote spermatagonial cells survive and improve sperm damage	[65]
ICR male mice	Spermatogenesis defects	hUC-MSC	Tail vein injection /2 × 106 cells	↑PCNA, SYCP3, MLH1, DMC1 ↑SOD1, CAT, PRDX, and BCL2 ↓BAX	Improves spermatogenic damage	[66]
SD male rats	Asthenospermia	hUC-MSC	Local testicular injection /3 × 104/side cells	↑p-AKT/AKT, p-mTOR/mTOR ↓LC3II/I, Beclin-1, LC3	Improve testicular damage and sperm quality	[67]
ICR male mice	Non-obstructive azoospermia	hUC-MSC-exo	Local testicular injection / 20 µg exosomes	↑vasa, miwi, Stra8, Dazl ↑BCL-2 ↓BAX, CASPASE-3	There was partial recovery of spermatogenesis	[68]
C57BL/6 male mice	Testicular injury	hUC-MSC-exo	Scrotal injection /1.5 × 109 exosomes	Inhibition of p38 lightning MAPK/ERK/AKT pathway Promote DNA damage repair	It can improve the morphology of testis, interstitial cells and seminiferous tubules, and increase the pregnancy rate	[69]
hSerC/C	Apoptosis	hUC-MSC-exo	Co-culture /1.50 × 109 exosomes	It promoted cell proliferation, DNA damage repair, and anti-apoptosis	Increased cell number and viability	[69]
C57BL/6 female mice	PCOS	hUC-MSC	Tail vein injection /2 × 106 cells	↓TNF-α, IL-1β, IFN-γ, M1 macrophages ↑IL-10, M2 macrophages	It can improve the morphology of polycystic ovary and estrous cycle disorder, and relieve the local inflammatory response of uterus	[73]
C57BL/6 female mice	POI	hUC-MSC	Intravenous / 1 × 106 cells	↓IL-6, IL-1β ↑IL-10, TSG-6, VEGF	Reduce the level of inflammatory infiltration in ovarian tissue	[74]
GCs	Apoptosis	hUC-MSC	Co-culture	↑Phosphorylated AKT and phosphorylated P38/total P38	The number of apoptotic cells was reduced	[74]
SD female rats	POI	3D hUC-MSC spheroids	Local ovarian injection /1 × 106 cells	↑GSH, P62 ↓MDA, ATG5, LC3A/B, BAX ↑BCL2, PCNA	Improve ovarian function and hormone secretion levels	[75]
Ovaries of ICR female mice	POI	hUC-MSC	In vitro embedding culture/cell volume of one 24-well	↓ Fe2+ ↑T-GSH, GSH, GPX4 ↓ACSL4 ↓COL-1,,α-SMA	It can improve ovarian function and reduce the degree of ovarian fibrosis in POI mice	[78]
ICR female mice	POI	hUC-MSC	Intravenous injection/1 × 106 cells	Activating the PI3K pathway ↑ Ability to metabolize lipids ↓ Monosaccharide concentration	Improve ovarian function	[79]
KGN	Cell damage	hUC-MSC-EV	Co-culture	Activation of PI3K/AKT signaling pathway	Inhibition of apoptosis	[80]
C57BL/6 female mice	POI	hUC-MSC-exo	Local ovarian injection/0.45 µg exosomes	↓ROS levels ↑NRF2, GPX4	Improved ovarian function	[81]
C57BL/6 female mice	POI	IGF-1-carrying hUC-MSC-EV	Tail vein injection /2 × 107 EVs	Activation of the Nrf2/HO-1 pathway ↓ROS, MDA, LC3II/I, Beclin-1 ↑GSH	Inhibiting excessive autophagy of GCs and improving ovarian function	[82]
KGN	Apoptosis	hUC-MSC-EV	Co-culture /30µg/ml EVs	↓LC3BII/I, Beclin-1, Parkin, PINK1 Through METTL3 inhibit PINK1/Parkin pathways	It can improve the autophagy of GCs and reduce the number of apoptosis cells	[83]
C57BL/6 female mice	PCOS	hUC-MSC-EV	Intraperitoneal injection /150µg EVs	↓METTL3, LC3, Beclin-1, Parkin, PINK1	Enhanced ovarian function in PCOS mice and others	[83]
C57BL/6 female mice	POI	hUC-MSC-exo	Tail vein injection / 150 µg exosome protein	↑AMPK, LC3BII/I ↓SQSTM1	Improve ovarian morphology and function	[84]
SD female rats	IUA	hUC-MSC	Tail vein injection /1 × 10 cells7 /kg	↑IL-10 ↓IFN-γ, TNF-α, IL-2 ↑VEGFA, MMP9, CD31 ↓α-SMA, TGF-β	It can alleviate endometrial fibrosis, promote the proliferation of endometrial cells, and improve the fertility of rats	[88]
SD female rats	Uterine scars	hUC-MSC	Muscle tissue on both sides of the uterine scar/5 × 105	↑CD206, CD163, IL-10 M1 to M2 polarization ↓iNOS, TNF-α ↑VEGF, CD31	Promote tissue regeneration and angiogenesis in scar area	[51]
ESC	Apoptosis	hUC-MSC-miR-455-5p mimics	Co-culture / 1 × 105 cells	miR − 455-5 p downgrade SOCS3 promote JAK2 and STAT3 phosphorylation	Promote the ESC proliferation and cell cycle progress	[89]
Female Mice	IUA	hUC-MSC-miR-455-5p mimics	Intraperitoneal injection /1 × 105 cells	miR-455-5p down-regulated SOCS3 and promoted the phosphorylation of JAK2 and STAT3	Reduce the endometrial fibrosis	[90]
hEndoSCs	Cell apoptosis	hUC-MSC-exo	Co-culture /96 Wells	↑Bcl-2 ↓Caspase-3, Cleaved Caspase-3 Activation of PTEN/AKT signaling pathway	Promotes cell proliferation	[90]
ESC	Cell damage	hUC-MSC-exo	Co-culture /24 Wells	↑miR-7162-3p Targeting the 3-UTR inhibits APOL6 expression	Inhibition of apoptosis	[91]
HESC	IUA	hUC-MSC-exo	Co-culture	↑miR-140-3p Targeting FOXP1 inhibits the TGF-β/Smad pathway ↓α-SMA, COL1A1, CTGF	HESC fibrosis induced by inhibition of TGF-β	[92]
SD female rats	IUA	hUC-MSC-exo	Local uterine injection /100µg exosomes	↑miR-202-3p Early ↓MMP11 ↑COL1A1, COL3A1, COLVI, and FN proteins	Early promotion of ECM addiction is conducive to wound healing	[93]

Combined application of hUC-MSCs and hUC-MSC-EVs with scaffolds/gels

After EVs successfully enter the uterine cavity, they must be effectively internalized by endometrial epithelial cells or stromal cells to exert biological effects. This process relies on highly specific matching between the surface ligands on EVs and the receptors on the endometrial cell surfaces. However, as EVs typically originate from non-uterine cells (MSCs), their surfaces often lack specific ligands that efficiently bind to endometrial cells, resulting in poor endocytic efficiency on the EVs. Irene et al. [94] demonstrated in a mouse model that following intrauterine infusion or tail vein injection, only 0.59% and 0.65% of transplanted cells, respectively, were detected in the perivascular regions of the endometrium. This limited cell homing capacity and short retention time severely restrict their clinical translational applications. Therefore, optimizing the delivery efficiency of stem cells and their derived EVs, enhancing their therapeutic potential, and maintaining their in vivo activity have become core challenges requiring urgent resolution in stem cell-based regenerative therapies. ( As shown in Fig. 4)

Fig. 4.

Combined application of hUCMSCs /EVs with biomaterials. The integration of hUC-MSCs and EVs with biomaterials significantly enhances the efficiency of hUC-MSCs and EVs utilization compared to their individual application. This approach more effectively facilitates cytokine secretion and ameliorates disease conditions.

(source of photo: Figdraw)

Combination of stent with hUC-MSC and hUC-MSC-exo

Porous scaffolds are essential components of tissue engineering materials that facilitate cell adhesion and the transport of nutrients and oxygen, thereby playing a pivotal role in tissue repair and regeneration. Among these, collagen scaffolds are particularly noted for their capacity to create an optimal microenvironment for cells by emulating the biochemical and structural characteristics of the natural ECM. Xin et al. [95] loaded 5 × 10⁵ hUC-MSCs onto a 2.5 cm×0.5 cm collagen scaffold (CS) and administered it via intraperitoneal injection to treat a rat model of endometrial injury. Tracking via immunofluorescence and histological staining revealed a significant presence of CS and hUC-MSCs three days post-transplantation, with numbers decreasing over time. Fifteen days after transplanting CS/hUC-MSCs into IUA rats, uterine examination revealed that the CS/hUC-MSC group exhibited only hemorrhage and edema at the uterine end compared with the control and CS groups. Histological analysis revealed distinct tubular structures and significantly increased endometrial thickness in the CS/hUC-MSC group possessed. As the number of days post-transplantation increased, the uterus progressively returned to a more normal state. This effect may arise because CS/hUC-MSC transplantation creates a favorable environment for tissue repair during the early stages of endometrial injury. Subsequently, through paracrine mechanisms, it elevates the levels of VEGF-A, TGF-β1, and Platelet-derived Growth Factor (PDGF-BB), thereby significantly inhibiting endometrial cell apoptosis and promoting proliferation. This mechanism is consistent with the therapeutic effects of hUC-MSCs. where VEGF enhances vascular permeability, TGF-β1 induces endometrial proliferation by modulating immune responses, and PDGF is correlated with stromal cell proliferation. Lu et al. [96] further investigated the mechanism by which CS/hUC-MSC constructs regenerate the damaged endometrium. They injected 10 × 10⁵ cells mixed with 25 µl (6.67 mg/ml) degradable collagen fibers into uterine scar marking points at 30 and 60 days. The findings revealed that the scaffold confined hUC-MSC dispersion within scar areas and promoted the long-term retention of hUC-MSCs in uterine scars. Compared to hUC-MSCs alone, CS/hUC-MSCs transplanted at 30 days exhibited more pronounced neovascularization and fewer visible polyps and contractures. By 60 days post-transplantation, macroscopically examined uterine scars treated with CS/hUC-MSCs resembled normal tissues. This may be attributed to the ability of the CS/hUC-MSC treatment group to significantly degrade collagen in uterine scars by upregulating MMP-9 expression, thereby promoting the regeneration of endometrial glands and myofibers, which effectively improved pregnancy outcomes in rats. The decellularized human amniotic membrane (AM) matrix is an immunologically safe biological scaffold. Compared to synthetic materials, it resembles the three germ layers and has been used for tissue repair for a long time. Wang et al. [97] seeded 1 × 10⁶ hUC-MSCs onto a 1 cm×2 cm AMM, which was then applied to the injured uterine site. Compared to the control group, the damaged endometrium exhibited more intact glandular structures, tighter luminal and glandular epithelial cells resembling normal cells, significantly increased endometrial thickness, and improved morphology. Significant increases in the mRNA and protein expression of keratin, integrin β3, and vimentin reflected enhanced growth of endometrial epithelial and stromal cells and improved endometrial receptivity in rats. This study also demonstrated that the hUC-MSC-AMM group reduced fibrosis by promoting the degradation of fibrous connective tissue through increased MMP9 expression, consistent with the findings of Lu et al. [96].

Xin et al. [98] further investigated the effects of scaffolds (2.5 cm×0.5 cm) loaded with 3 × 10¹⁰ hUC-MSC-exo on endometrial regeneration and recovery. Consistent with Xin’s findings [95], CS/hUC-MSC-exo levels decreased over time in the endometrium, and uterine morphology tended to normalize by day 60 post-transplantation. Furthermore, they discovered that CS/exosomes enhanced the expression of M2 macrophage-associated cytokines, including IL-10, TGF-β1, and VEGFB, via 21 miRNAs. This mechanism promotes M2 macrophage polarization in the late stage, reduces inflammation, and amplifies the anti-inflammatory response of the body. Consequently, it facilitates endometrial regeneration, collagen remodeling and fertility restoration.

Studies have shown that biomaterials help hUC-MSCs and their exosomes remain in their intended locations. These materials also work well for fixing damaged endometrial tissue because of their natural healing properties.

Combined application of hydrogels and hUC-MSCs

Hydrogels are soft, semi-solid substances with three-dimensional polymeric networks. This structure enables hydrogels to retain large amounts of water and aqueous solutions in their pores. This property grants hydrogels the ability to carry hydrophilic therapeutic agents and mimic tissue microenvironments, offering new directions for disease treatment [99]. Although scaffolds have garnered extensive attention for their unique properties, injectable hydrogels meet practical clinical demands by enabling minimally invasive procedures that significantly reduce infection risks and recovery times, making them the preferred choice for treating IUA [100]. Numerous methods exist for preparing injectable hydrogels, with in situ gelation typically relying on ongoing crosslinking processes to induce a gradual sol-gel transition of the precursor solution at the injection site. In recent years, diverse chemical and physical crosslinking mechanisms have been employed to develop in situ gelation hydrogels, including click chemistry, enzyme-catalyzed crosslinking, and stereocomplexation [101]. Hu et al. [102] prepared an injectable hyaluronic acid hydrogel via a Diels-Alder reaction using maleimide and methylfuran-modified hyaluronic acid. The hydrogel was loaded with hUC-MSCs (1 × 10⁷ cells/mL) and injected into the injured uterine horns. They found that post-injection, the fluid not only permeated the tissue comprehensively, adapting to the complex geometry of the uterine cavity, but also served as a mechanical barrier isolating the injured area and as a reservoir for cytokines and bioactive mediators, enabling the sustained release of therapeutic paracrine factors. Both in vitro and in vivo studies demonstrated the therapeutic efficacy of hUC-MSC-loaded injectable hydrogels in the damaged endometrium. In vitro co-culture experiments revealed that the hydrogel enhanced cell proliferation, migration, angiogenesis, and anti-fibrotic capacity in both HESCs and HUVECs. In vivo experiments further demonstrated that the hUC-MSC-embedded hydrogel-loaded treatment group exhibited more endometrial glands and a smaller fibrotic area than the hUC-MSC-only treatment group. This effect may have resulted from hydrogel-enhanced hUC-MSC retention in vivo, thereby promoting VEGF expression and the polarization of macrophages from the M1 to M2 phenotype more effectively. Pluronic F-127 is an injectable hydrogels. Zhou et al. [103] injected (20%) Pluronic F-127 hydrogel-encapsulated 5 × 10⁶ hUC-MSCsinto the right uterine horn of IUA rats. In the treatment group with Pluronic F-127-loaded hUC-MSCs, HE staining revealed a significant increase in the number of endometrial glands. This effect may arise from the increased expression of Von Willebrand Factor, Vegfa, EGF and HGF under the stimulation of the inflammatory cytokine IL-1β, thereby promoting angiogenesis and endometrial cell proliferation. This suggests that one mechanism underlying the therapeutic action of hUC-MSCs involves stimulation by inflammatory cytokines at the injury sites. Zhang et al. [104], consistent with the findings of Hu et al. [102], prepared an injectable hydrogel based on oxidized hyaluronic acid and hydrazide-grafted gelatin loaded with 5 × 10⁶ hUC-MSCs. They observed significantly greater endometrial thickness, glandular number, and vascular restoration in the treatment group compared to the control group. This effect may stem from the hUC-MSC-loaded hydrogel therapy modulating endometrial VEGF expression via the Mitogen-activated protein kinase kinase (MEK)/ERK1/2 signaling pathway and regulating inflammatory factor balance. Furthermore, they demonstrated in a rat model of endometrial injury that hydrogel-loaded hUC-MSCs promoted endometrial repair by enhancing vascularization and the ERK1/2 signaling pathway to promote VEGF expression in the endometrium and regulate inflammatory factor balance. Furthermore, they demonstrated that injecting hUC-MSC-loaded hydrogels into a rat model of endometrial injury significantly restored embryo implantation and live birth rates. Notably, no adverse effects were observed in maternal rats or in fetal and offspring development. These studies revealed that the local inflammatory environment can promote tissue repair by stimulating hUC-MSCs to secrete cytokines. Simultaneously, hUC-MSCs regulate the balance of inflammatory mediators, ultimately improving fertility in mice.

In summary, the therapeutic mechanism of injectable hydrogels loaded with hUC-MSCs aligns with that of stem cells, but the unique properties of the hydrogel enhance hUC-MSC proliferation and reduce migration time. This characteristic significantly improves hUC-MSC survival and therapeutic efficacy in vivo. Additionally, researchers combined 1 × 10⁵ hUC-MSCs with hyaluronic acid to treat a POI mouse model, demonstrating effective improvements in ovarian morphology, cell proliferation, angiogenesis, and fertility. Furthermore, cytokine array analysis demonstrated significantly upregulated cytokine secretion in the matrix of hydrogel-treated hUC-MSCs [105], indicating that the hydrogel not only promotes drug retention but also enhances its biological activity. This combined therapy holds promise for clinical application as a potential future treatment strategy for damaged reproductive organs.

Clinical application

In conclusion, both hUC-MSCs and hUC-MSC-EVs have been extensively utilized in the treatment of reproductive system disorders, primarily because of their low immunogenicity and ease of procurement. To date, over 8,500 clinical studies involving stem cell products have been performed. Table 2 summarizes the ongoing studies involving hUC-MSCs that are currently registered in the ClinicalTrials.gov database, with results that are yet to be reported. Nevertheless, similar to other cell therapies, hUC-MSCs present several potential risks to patients. These include short-term risks, such as infections, embolism, or acute immunogenic reactions, as well as long-term concerns related to chronic immunogenicity and tumorigenicity. Table 3 summarizes the completed clinical trials of hUC-MSCs and their EVs for the treatment of reproductive disorders. The table evaluates the feasibility, efficacy, and adverse reactions associated with the clinical application of hUC-MSCs and hUC-MSC-EVs.

Table 2.

Registered ongoing studies

Provider	Phase	Primary endpoints	Status	Key outcomes	ID
Erectile dysfunction	Ⅰ, Ⅱ	Penile Doppler ultrasonography for assessment of erectile function	Not yet recruiting	/	NCT06550752
PCOS	Ⅰ, Ⅱ	Parameter in testing the cytokine/adipokine/hormone profile Insulin, Glucose Plasma, and Insulin Resistance	Unknown status	/	NCT05279768
POI	Not applicable	Follicular development rate, Changes in blood flow index in the ovary, Clinical pregnancy rate	Unknown status	/	NCT05308342
POI	Ⅰ, Ⅱ	Number of said follicle, Number of antral follicle development, Ovarian volume, Pregnancy rate	Unknown status	/	NCT03033277

Table 3.

Experiments completed

Indication	Product	Route/dose	Follow-up	Outcomes	Adverse events	Ref.
POI	hUC-MSC	Ovaries /0.5 × 10 7cells n = 61 for the first inoculation n = 50 for the second inoculation The second vaccination n = 30	After 6 months, hormone levels and follicle growth were checked on the third day of the menstrual cycle. Ultrasound was performed after treatment.	Patients of follicular, AMH levels rise, and show better in patients undergoing multiple doses of ovarian function 61 cases, 1 case was naturally conceived, ICSI fertilization embryo (3 cases)	No serious side effects or complications related to the treatment were observed	[108]
POI	CS/hUC-MSC	Ovaries/unilateral 5 × 106 cells injection on both sides, were transplanted 4 times	7 days interval measurement of serum estradiol, monitoring follicle development situation and ovarian area, for 3 months, once every two weeks, after at least a year	FSH group decreased significantly, the higher H2, ovarian volume increased, 8 cases, the increase in the number of mature follicle naturally conceived in 2 cases	Show no serious side effects or complications associated with therapy	[110]
IUA	hUC-MSC	Transferred into the uterine cavity /2 × 107 cells transplanted twice	Menstrual volume and duration were recorded, and the volumes of cesarean scar diverticulum and uterus were measured	Four patients by blood within three months a slight increase in late sixth endometrial thickness and uterine incision diverticulum, uterine cavity volume had a tendency to improve	Show no serious side effects or complications associated with therapy	[111]
IUA	CS/hUC-MSC	Implantation into the uterine lumen /1 × 107 cells	Three months after surgery endometrial thickness and blood flow measurement, hysteroscopy examination evaluate IUA score and anatomical structure of uterine cavity, normal uterine cavity preparation embryo transfer or pregnancy, and follow-up to the end of pregnancy	4 cases were not caused by intrauterine adhesions after treatment Mild adhesions were observed in 6 patients Ten patients recovered from severe to moderate AS Five patients did not improve 10 patients with pregnancy, 8 cases of live births, 1 case of premature birth, 1 case in late pregnancy	No inflammatory reaction was detected by endometrial biopsy 3 months after surgery, and no placental complications were noted	[112]
Asherman syndrome	CS/hUC-MSC	Attached to uterine wall /2 × 107 cells Transplant in two consecutive menstrual cycles	ET, endometrial morphology, uterine receptivity, pregnancy outcome and endometrial histology were measured before and 3 months after surgery	Scar area is reduced, and the receptivity of endometrium, promoting angiogenesis, endometrial glands and the increase in the number of 17 cases, 15 cases of patients accepted FET, 3 cases of pregnancy, spontaneous abortion in 1 case, natural pregnancy in 1 case	Show no serious side effects or complications associated with therapy, found no placental complications	[113]
Refractory thin endometrium	CS/UC-MSC	Attached to uterine wall /2 × 107 cells	Physical examination, 2D ultrasound scan, and psychological evaluation were performed at 1 month 3D ultrasound scan, laboratory tests, and ECG were performed at 3 months Pregnancy outcomes as well as obstetric and perinatal complications were carefully recorded	Compared with the control group (saline /CS), the uterine cavity structure was better, the endometrial receptivity and embryo implantation rate were higher, and the cumulative live birth rate was not significantly increased Forced symptoms of fear and anxiety scores significantly lower than before treatment score after treatment	Showed no serious side effects or complications associated with therapy, obstetric and perinatal adverse reactions	[109]

Currently, three primary methods are used for the administration of pharmaceuticals in the treatment of female reproductive disorders: vaginal, intrauterine, and ovarian. Vaginal administration is the most prevalent and minimally invasive method. Pharmaceuticals are absorbed through the vaginal mucosa, allowing direct action on the local vaginal and cervical regions [106]. Marina’s research indicates that during local vaginal administration, cells predominantly accumulate at the injection site and do not effectively diffuse throughout the pelvic organs [107]. In contrast, intrauterine and ovarian administration methods promote the direct and efficient homing of transplanted cells to the target tissues; however, this method requires physician intervention, making direct injection the preferred clinical approach for treating uterine and ovarian diseases with MSCs [108, 109]. Nevertheless, if EVs can be engineered or chemically conjugated to carry ligands or antibodies that specifically recognize diseased tissues, such as the uterus or ovaries, this would significantly enhance their therapeutic efficacy and potential for non-invasive delivery.

Outlook

Recent studies have highlighted the effects of hUC-MSCs and EVs in the treatment of reproductive disorders. However, translating these findings into clinical practice poses significant challenges to researchers. This discussion focuses on safety considerations of MSCs administered via in vivo infusion: (1) Pulmonary First-Pass Effect: This occurs when MSCs, introduced through peripheral venous infusion, become trapped in lungs during initial passage due to their large size. Over hours or days, these cells are cleared from the pulmonary system and redistributed to inflammation sites. Research suggests that MSCs may remain in the lungs for up to 150 days, thereby reducing the number of cells available for therapeutic purposes. Although arterial administration may reduce mechanical entrapment in the lungs, larger MSCs may still pose a risk of vascular occlusion, potentially causing ischemia or organ infarction [114, 115]. (2) Batch Variability: Aging in donors and recipients affects MSC biodistribution. In elderly donors and recipients, MSC transplantation efficiency is reduced, and the harvested MSCs show decreased proliferation and differentiation capacities. This variability results in differences in the content and quantity of EVs released by cells [116]. (3) Tumor-Promoting Effects: Given MSCs’ ability of MSCs to home to tumor sites, there is a potential risk for therapeutic MSCs to transform into cancer-associated MSCs. Zhuang et al. [117]. conducted a comprehensive review of the effects of MSCs on various tumor cell types, concluding that hUC-MSCs tend to promote breast cancer progression rather than inhibit it. Further research has demonstrated that hUC-MSCs can enhance the proliferation and migration of basal cell carcinoma by activating ERK signaling. Additionally, EVs derived from hUC-MSCs have been shown to increase the proliferation of lung adenocarcinoma tumor cells and decrease apoptosis through the transfer of microRNA-410. Notably, EVs overcome certain intrinsic limitations associated with stem cell therapies owing to their inability to replicate themselves. Nevertheless, the production process necessitates large-scale amplification while maintaining high batch-to-batch consistency and economic feasibility in compliance with GMP standards. Given that EV yield, composition, and bioactivity are substantially affected by upstream and downstream processing decisions—such as cell passage, cell density, and EV harvesting frequency—it is imperative to balance cellular requirements with engineered process control parameters and the cellular environment [118, 119].(1) To obtain GMP-compliant EVs, donors must undergo rigorous screening to eliminate the potential risk of infectious or genetic diseases. The production environment must maintain strict sterility to prevent microbial contamination [120, 121]. (2) No consensus exists on EVs separation techniques. For instance, size-exclusion chromatography can partially separate NVEPs from EVs based on particle size differences; however, it cannot entirely exclude the co-purification of NVEPs, resulting in a mixture of multiple particles. Furthermore, commercial exosome purification kits based on polymer precipitation are easy to use but lack specificity. They not only struggle to yield highly purified exosome subpopulations but also fail to effectively distinguish between broadly defined EVs and EP, ultimately yielding a mixture of various extracellular components [22]. (3) Post-preparation EVs require proper formulation and storage under suitable conditions to prevent the loss of physical integrity and biological activity of EVs. Improper storage significantly affects particle concentration and internal stability, further diminishing cellular uptake efficiency and in vivo biodistribution. Research indicates that ultra-low temperature storage at -80 °C is currently recognized as the optimal condition for maintaining EV stability. However, this approach faces significant limitations in terms of equipment costs, energy consumption, and cold chain logistics, posing severe challenges to large-scale clinical applications and their economic viability. Consequently, freeze-drying technology has garnered widespread attention as a viable long-term food preservation strategy. By removing water to inhibit degradation reactions, it offers a potential alternative for the stable storage and convenient transportation of EVs [122].

As the therapeutic applications of exosomes continue to expand, the European Network for the Role of Microvesicles and Exosomes in Health and Disease, in conjunction with the International Society for Extracellular Vesicles, has issued consensus statements and recommendations to actively promote the standardized clinical use of exosomes. These guidelines delineate the Standard Operating Procedures to be adhered to throughout the entire process of isolating, identifying, processing, quality controlling, and producing clinical-grade exosomes, with the objective of ensuring their safety, reproducibility, and efficacy. These standardized frameworks establish a crucial regulatory and scientific basis for the evidence-based and rational clinical application of exosomes [123]. Consequently, even the most recently developed and innovative human therapeutic products derived from exosomes face substantial challenges across various dimensions, including manufacturing consistency, functional characterization, efficacy evaluation, and regulatory compliance issues.

Abbreviations

MSCs

Mesenchymal stem cells

EVs

Extracellular vesicles

BM-MSCs

Bone marrow mesenchymal stem cells

AD-MSCs

Adipose-derived mesenchymal stem cells

hUC-MSCs

Human umbilical cord mesenchymal stem cells

NVEP

Non-vesicular extracellular particles

MIXL1

Mix Paired-Like Homeobox

SDF-1/ CXCL12

Stromal cell-derived factor-1

CXCR4

C-X-C motif chemokine receptors 4

VLA-4

Very late antigen-4

MMPs

Matrix metalloproteinases

TGF-β1

Transforming Growth Factor-β

EGF

Epidermal Growth Factor

PGE2

Prostaglandin E2

IDO

Indoleamine 2,3-dioxygenase

HGF

Hepatocyte growth factor

FGF-2

Fibroblast growth factor-2

NGF

Nerve growth factor

mTCs

Mouse testicular cells

Oct4

POU Class 5 Homeobox 1

Vasa/Ddx4

DEAD-Box Helicase 4

Dazl

Deleted In Azoospermia Like

Stra8

Stimulated By Retinoic Acid Gene 8

Scp3

Synaptonemal complex protein

Miwi

Piwi-like protein 1

TNF-α

Tumor Necrosis Factor

CM

Conditioned medium

HUVECs

Human umbilical vein endothelial cells

PCNA

Proliferating Cell Nuclear Antigen

SYCP3

Synaptonemal Complex Protein 3

MLH1

MutL Homolog 1

DMC1

DNA Meiotic Recombinase 1

SOD1

Superoxide Dismutase 1

CAT

Catalase

PRDX

Peroxiredoxin

BCL-2

BCL-2 Apoptosis Regulator

BAX

BCL-2 Associated X, Apoptosis Regulator

NRF2

Nuclear factor erythroid 2-related factor 2

PI3K

Phosphoinositide 3-kinase

AKT

Protein kinase B

mTOR

Mammalian target of rapamycin

LC3

Light chain 3

Beclin-1

BECN1

hSerCs

Human testicular supporting cells

p38 MAPK

p38 mitogen-activated protein kinases

ERK

Extracellular signal-regulated kinases

POI

Premature Ovarian Insufficiency

PCOS

Polycystic Ovary Syndrome

DHEA

Dehydroepiandrosterone

IFN-γ

Interferon-gamma

VEGF

Vascular endothelial growth factor

ROS

Reactive oxygen species

FSH

Follicle-stimulating hormone

E2

Estradiol

MDA

Malonic dialdehyde

P62

SQSTM1

ATG5

Autophagy Related 5

GSH

Glutathione

GPX4

Glutathione Peroxidase 4

ACSL4

Acyl-CoA synthetase long chain family member 4

α-SMA

α-smooth muscle actin

MDA

Malonic dialdehyde

ATG5

Autophagy related 5

CTX

Cyclophosphamide

KGN

Human ovarian granulosa cells

HO-1

Heme oxygenase-1

Parkin

Parkin RBR E3 ubiquitin-protein ligase

PINK1

PTEN-induced kinase-1

METTL3

Methyltransferase-like 3

AMPK

Adenosine 5‘-monophosphate (AMP)-activated protein kinase

IUA

Intrauterine adhesions

ECM

Extracellular matrix

iNOS

Inducible nitric oxide synthase

CD206

Mannose Receptor C-Type 1

CD31

Platelet and Endothelial Cell Adhesion Molecule 1

JAK2

Janus Kinase 2

STAT3

Signal Transducer and Activator of Transcription 3

ESCs

Endometrial stromal cells

SOCS3

Suppressor of Cytokine Signaling 3

PTEN

Phosphatase and Tensin Homolog

hEndoSCs

Human endometrial stromal cells

FOXP1

Forkhead box protein P1

HESC

Human endometrial stromal cells

COL1A1

Collagen Type I Alpha 1 chain

CTGF

Cellular Communication Network Factor 2

TGF-β

Transforming growth factor-β

CS

Collagen scaffold

AM

Amniotic membrane

PDGF-BB

Platelet-derived Growth Factor

MEK

Mitogen-activated protein kinase kinase

MIXL1

Mix Paired-Like Homeobox

Author contributions

Xin Guo and Bingchun Liu conceived and wrote the article. Peixin Xu, Hong Chen, Jing Gao and Dongmei Yao, Xin Li produced the figures and revised the manuscript. Yurong Wang, Tong Wang, Hongrui Yao and Shuwei Qiao produced the tables. Jile Huge and Jianlong Yuan reviewed and edited the manuscript and obtained financial support.

Funding

National Natural Science Foundation of China (31960154);Key Research and Development and Achievement Transformation Program of Inner Mongolia Autonomous Region(2025YFSH0093); Inner Mongolia Science and Technology Project (2025YFSH0089); Inner Mongolia Autonomous Region Provincial Health Commission, Science and Technology Program for Building High-level Clinical Specialties in Public Hospitals in the Capital Region (2024SGGZ071); Science and Technology Project of the Scientific Research Joint Fund for Public Hospitals in Inner Mongolia Autonomous Region (2024GLLH0321); Inner Mongolia Medical University Affiliated Hospital Talent Training Project-Sailing Series; Youth Innovation Talents Training Program of the Inner Mongolia Autonomous Region“Prairie excellence” Project (Q2022085); Natural Science Foundation of Inner Mongolia (2023QN03047); Central Guided Local Science and Technology Development Funds Project (2023ZY0004); Affiliated Hospital of Inner Mongolia Medical University National Nature Cultivation Programme (2023NYFYPY006); 2024 Joint Fund for Research in Public Hospitals Science and Technology Projects (2024GLLH0298).

Data availability

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors of this manuscript consent to publication.

Competing interests

The authors declare no competing interests.