Advances in human umbilical cord mesenchymal stem cells-derived extracellular vesicles and biomaterial assemblies for endometrial injury treatment

Abstract

Endometrial injury caused by repeated uterine procedures, infections, inflammation, or uterine artery dysfunction can deplete endometrial stem/progenitor cells and impair regeneration, thereby diminishing endometrial receptivity and evidently lowering the live birth, clinical pregnancy, and embryo implantation rates. Currently, safe and effective clinical treatment methods or gene-targeted therapies are unavailable, especially for severe endometrial injury. Umbilical cord mesenchymal stem cells and their extracellular vesicles are characterized by their simple collection, rapid proliferation, low immunogenicity, and tumorigenicity, along with their involvement in regulating angiogenesis, immune response, cell apoptosis and proliferation, inflammatory response, and fibrosis, Therefore, these cells and vesicles hold broad potential for application in endometrial repair. This article reviewed recent research on human umbilical cord mesenchymal stem cells as well as their extracellular vesicles in repairing endometrial injury.

Key Words: Endometrial injury; Umbilical cord mesenchymal stem cells; Extracellular vesicles; MicroRNA; Biomaterial assemblies; Regenerative repair

Core Tip: This study explored the therapeutic potential of human umbilical cord mesenchymal stem cells (hucMSCs) as well as their extracellular vesicles in repairing endometrial injury. We demonstrated that hucMSCs promote endometrial regeneration through various mechanisms, such as cell proliferation, anti-fibrosis, angiogenesis, and immunomodulation. The findings highlighted the advantages of hucMSCs, including their low immunogenicity and ethical acceptability, making them a promising candidate for clinical applications. Our work addressed current challenges in hucMSC therapy, laying the groundwork for future standardized protocols and potentially offering new hope for females with endometrial dysfunction.

INTRODUCTION

The synchronous development of high-quality embryos and normal endometrial receptivity is a crucial factor for successful pregnancies[1]. Endometrial injury severely affects endometrial receptivity, hindering embryo implantation and development, thereby leading to infertility and adverse perinatal outcomes. Currently, a gold-standard treatment is lacking, with most treatments failing to restore the regenerative characteristics of the endometrium.

As an early undifferentiated cell type, stem cells possess self-replication, high proliferation, and multidirectional differentiation capabilities[2] essential for endometrial regeneration[3]. Among them, human umbilical cord mesenchymal stem cells (hucMSCs), sourced from residual blood and discarded umbilical cord tissue after fetal cord ligation, participate in anti-inflammation, angiogenesis, immunomodulation, anti-apoptosis, and anti-fibrosis[4,5]. Compared to other stem cell sources, hucMSCs offer multiple advantages, including simple collection, rapid proliferation, low immunogenicity and tumorigenicity, and few ethical disputes.

Recently, hucMSCs have emerged as a research hotspot in reproductive medicine, especially for endometrial injury repair[6]. HucMSC-derived extracellular vesicles (hucMSC-EVs) are important communication carriers between stem cells[7]. Among the various molecules contained in EVs, microRNAs (miRNAs) are inseparably linked to tissue injury, repair, and regeneration[8] and have received more attention in endometrial repair and regeneration.

Despite numerous basic and clinical studies over the years investigating stem cells from various sources to promote endometrial regeneration and repair, little is known about the communication mechanism and regulatory role between hucMSCs and injured endometrial cells. A summary was conducted in this review on the regulatory mechanisms of endometrial regeneration, the pathophysiological characteristics of endometrial injury, and the existing data on hucMSC and hucMSC-EV treatment of endometrial injury repair, aiming to provide a scientific basis and reference significance for endometrial repair by hucMSCs.

ENDOMETRIAL DYNAMICS

The endometrium undergoes changes regulated by estrogen and progesterone throughout the female reproductive cycle, controlling inflammatory, anti-inflammatory, and epigenetic signaling pathways. Over a female’s lifespan, 400-500 cyclical proliferation, differentiation, shedding, repair, and regeneration processes occur[9]. At the cellular level, the endometrium contains epithelial cells (glandular epithelia and luminal epithelia), stromal cells, immune cells, and blood vessels. At the tissue level, the endometrium comprises two layers: (1) The functional layer, containing epithelial cells, loose stroma, and spiral arteries, is susceptibility to sex hormones. The functional layer has a thickness of roughly 1 mm during the early proliferative phase, reaches 8-16 mm at ovulation, and is shed during the menstrual period. The luminal epithelia in the functional layer contain cilia that aid in clearing cell secretions, assist in sperm transport, and promote embryo implantation, thereby contributing significantly to determining endometrial receptivity[10]; and (2) The basal layer includes adenocarcinoma cells, dense stroma, and short straight arteries, with no sensitivity to sex hormones. Its thickness is approximately 1 mm, exhibiting strong proliferation and repair abilities. During the menstrual period, the basal layer is shed and subsequently forms a new functional layer[11] (Figure 1).

Figure 1 Mesenchymal stem cells and damaged endometrium. This figure illustrates the key structures of the endometrium, including the functional and basal layers, and the types of stem cells (epithelial-like and stromal-like stem cells) involved in the repair process following injury. The main sources of mesenchymal stem cells and their contribution to endometrial repair were also highlighted.

In the menstrual cycle, the endometrium undergoes repeated injury and scarless repair, demonstrating a high degree of dynamic regenerative capacity. Therefore, in 1978, Prianishnikov[12] first proposed the possible existence of endometrial stem/progenitor cells. In 2004, Chan et al[13] were the first to discover, identify, and purify a small number of stem cells with high cloning activity and multilineage differentiation potential in human total hysterectomy specimens. In 2006, the presence of epithelial stem cells and stromal stem cells within rodent endometrium was again traced by Chan and Gargett[14]. They suggested that this result was related to endometrial regeneration. Subsequently, numerous basic and clinical studies have demonstrated that endometrial stem/progenitor cells not only coexist in the endometrial functional and basal layers but also arise from bone marrow-derived stem cells in the circulatory system[15]. Such a regenerative process occurs not only in contexts, such as childbirth, endometrial resection, and menopause hormone therapy, but also in the menstrual cycle[16]. These findings highlighted the presence of endometrial stem/progenitor cells from varying sources and tissues, each contributing to the repair of various types of endometrial injuries, such as thinning, atrophy, and dysfunction.

ENDOMETRIAL INJURY

The injured endometrial basal layer may result in endometrial fibrosis, a decrease in gland number, and invasion by inflammatory factors, thereby leading to excessive consumption of endometrial stem/progenitor cells and/or endometrial regeneration disorder. Among them, the injured endometrial basal layer can be caused by artificial abortion, diagnostic curettage, repeated manipulation of endometrial cavity, tuberculosis infection, and uterine artery embolization (Figure 1). The pathophysiological characteristics of endometrial injury include increased uterine artery blood flow resistance, poor vascular development, slow growth of glandular epithelium, endometrial fibrosis, and a decline in gland numbers, resulting in ischemia and hypoxia of endometrial cells and an obstacle to tissue regeneration[17].

Clinically, endometrial injury is manifested as thin endometrium or intrauterine adhesions (IUA), with its pathogenesis remaining unclear. Additionally, the related hypotheses are composed of neural reflex theory, abnormal regulation of signaling pathways, fibrosis hyperplasia theory, changes in the uterine microenvironment and fibrosis, abnormal differentiation of stem cells, and inflammatory responses caused by adhesive fibroblasts. Confirmed signaling pathways or factors mainly include transforming growth factor-β (TGF-β)/small mothers against decapentaplegic (Smad)[18], Wnt/β catenin[19], nuclear factor-κB signaling pathway[20], estrogen receptor-α, cellular communication network factor 2, matrix metalloproteinase-9[21], and ΔNp63[22].

Thin endometrium markedly declined embryo implantation rate, live birth rate, and clinical pregnancy rate, and its occurrence is linked to repeated implantation failure, recurrent miscarriage, and infertility[23], with an incidence rate of 2.4%-8.5%[24]. However, no consensus has been reached regarding thin endometrium. An endometrial thickness of < 8 mm on trigger day and < 7 mm in the frozen-thaw embryo transfer cycle were defined as thin endometrium, as proposed by the Canadian Fertility and Andrology Society guideline in 2019[25]. In 2019, the European Society of Human Reproduction and Embryology recommended informing patients of the risk of reduced pregnancy rate when endometrial thickness is < 8 mm[26]. IUA refers to damage, either partial or complete, to the endometrial basal layer. This can cause local adhesions in the endometrial cavity, resulting in endometrial fibrosis and atrophy, thereby reducing endometrial receptivity. The clinical manifestations include decreased menstrual blood, prolonged menstrual periods, dysmenorrhea, amenorrhea, recurrent miscarriage, and infertility.

Endometrial repair includes endometrial epithelial regeneration, vascular repair, proteolytic enzyme action, and growth factor stimulation. The repair mechanisms are still unclear. Currently, three hypotheses are proposed: Surviving endometrial epithelial proliferation and differentiation; endometrial stem cell differentiation; and epithelial-mesenchymal transition (EMT). Clinical treatment methods consist of hysteroscopic adhesion resection, use of high-dose estrogen, oral medications to improve endometrial blood supply (such as low-dose aspirin, sildenafil citrate, and pentyl caffeate), intrauterine infusion of cytokines (such as platelet-rich plasma, growth hormone, and granulocyte colony-stimulating factor), and stem cell therapy[27]. However, hysteroscopic adhesion resection is ineffective for severe IUA, with a recurrence rate as high as 62.5% post-surgery[28]. Long-term high-dose estrogen use possibly enhances the risk of thrombosis and abnormal endometrial hyperplasia or carcinogenesis. The clinical efficacy of medications like low-dose aspirin remains uncertain. Intrauterine infusions of cytokines lack unified protocols and safety evaluations, and their efficacy in treating severe injuries is poor. Therefore, there is an urgent need for a safe, effective, and ethically acceptable treatment for endometrial injury. Recent studies have increasingly highlighted the critical role of stem cells, particularly MSCs, in the repair and regeneration of endometrial injury, and its role has garnered growing attention[29,30].

REPAIR OF ENDOMETRIAL INJURY BY HUCMSCS

HucMSCs and EVs

As a cell type that exists in an undifferentiated form, stem cells possess strong self-renewal, differentiation, and proliferation abilities. Based on their source, stem cells can be allocated into adult and embryonic stem cells. Stem cells were first isolated from the mouse blastocyst in 1981[31]. Under physiological conditions, stem cells participate in the growth, development, and homeostasis of tissues, organs, and whole systems. Under pathological conditions, stem cells regulate disease progression and tissue repair. Currently, stem cells have been applied in various fields, such as the production of stem cell-derived biomaterials for human tissues, the treatment of genetic diseases, regenerative medicine, and pharmacogenomics[32]. In 2007, the first report on stem cells targeting and promoting endometrial regeneration was published[33]. Additionally, stem cells have achieved significant outcomes in treating various infertility and reproductive system diseases, such as premature ovarian insufficiency, IUA, endometrial atrophy, pelvic organ prolapse, or endometriosis[34].

MSCs are adult stem cells originating from the embryonic early mesoderm layer, named for their ability to differentiate into interstitial cells. MSCs are isolatable and purifiable from several tissues, such as umbilical cord, bone marrow, neural tissue, fat, placenta, salivary gland, amniotic fluid, menstrual blood, and dental pulp[35,36]. Currently, MSCs used for endometrial regeneration mainly include adipose tissue-derived MSCs, menstrual blood-derived MSCs, bone marrow MSCs, and hucMSCs (Table 1). In 2011, Nagori et al[37] first reported injecting autologous bone marrow stem cells into the uterine cavity of patients with severe IUA, resulting in endometrial growth up to 8 mm and achieving clinical pregnancy through in vitro fertilization and embryo transfer.

Table 1 Summary of different mesenchymal stem cell types in endometrial injury treatment.

MSC type	Source	Mechanisms in endometrial repair	Key findings in endometrial injury studies	Ref.
Umbilical cord-derived MSCs	Endometrium	Proliferation, anti-fibrosis, angiogenesis	Support for endometrial regeneration by differentiating into functional endometrial cells	[85]
Bone marrow-derived MSCs	Bone marrow	Anti-inflammation, immunomodulation	Reduction of fibrosis and improvement in endometrial thickness and vascularization in preclinical models	[86]
Adipose tissue-derived MSCs	Adipose tissues	Paracrine signaling, anti-apoptosis	Significant potential in mitigating fibrosis and enhancing cellular regeneration	[87]
Amniotic MSCs	Amniotic membranes	Immunomodulation, anti-fibrosis	Success in reducing inflammation and supporting tissue repair of the endometrium in experimental settings	[88]
Menstrual blood-derived MSCs	Menstrual blood	Angiogenesis, epithelial-mesenchymal transition	Known for high proliferative potential and efficacy in improving endometrial receptivity and vascular repair	[89]

MSC: Mesenchymal stem cell.

HucMSCs are an MSC type derived from the residual blood and discarded umbilical cord tissue after umbilical cord ligation. They can be obtained from the entire umbilical cord (which may contain other cells) or separated and cultured from distinct compartments of the umbilical cord, including Wharton’s jelly MSCs, subamnion MSCs, amniotic membrane MSCs, and perivascular MSCs[38,39]. HucMSCs and their blood-derived progeny were previously regarded as medical waste. However, a study revealed that hucMSCs express low or no major histocompatibility complex I and II, along with low tumorigenicity and immunogenicity, positioning them as a crucial source of stem cells for immune rejection-free allogeneic transplantation[40]. Additionally, due to the non-invasive collection method and no ethical issues, hucMSCs are preferred for autologous and allogeneic transplantation compared to other sources of MSCs. They have been extensively studied and applied in tissue engineering and regenerative medicine, including reproductive medicine. However, the mechanisms of hucMSCs repairing injured endometrium are still unclear, and their long-term safety and application are still controversial. The majority of developed therapeutic methods remain in the experimental stage.

As research on MSCs advances, the therapeutic effects of MSCs are likely achieved through a large number of bioactive cytokines produced by their autocrine and paracrine secretion. These cytokines are secreted into the extracellular space, where they may be sorted into extracellular vesicles, including apoptotic bodies and microsomes, which can mediate intercellular communication.

Within this context, EVs have become a research hotspot resulting from their small size, low antigenicity and tumorigenicity, stable and easy-to-store content, non-invasive collection, and ease of large-scale production. EVs possess a lipid double-layer membrane structure, with a density of 1.09-1.18 g/mL and a diameter of 30-200 nm. They carry various nucleic acids, proteins, and lipids (including miRNA, long non-coding RNA, circular RNA, and messenger RNA), making them the main regulatory factors for cell-to-cell signaling. Current research has shown that in various injury repair models, hucMSC-EVs can promote angiogenesis, inhibit fibrosis, and facilitate collagen deposition through their autocrine and paracrine actions, even playing a more crucial role than MSCs themselves. To date, research on EV therapy has been reported, including reducing infarct size of myocardial infarction[41], alleviating liver fibrosis[42], promoting regenerative repair after liver injury[43], treating autoimmune demyelination[44], enhancing regenerative repair after skin injury[45], delaying the graft-vs-host disease onset[46], rescuing the bone marrow MSC function in lupus[47], protecting ischemic myocardium from ischemia/reperfusion injury[48], stimulating chondrocyte migration and proliferation in osteoarthritis[49], inhibiting tumor progression and angiogenesis[50], and promoting the dormancy of breast cancer metastatic lesions[51]. EVs have attracted considerable research interest as an alternative to traditional MSC-based cell therapy.

Repair mechanism research

Increasing research indicates that hucMSCs and hucMSC-EVs can be directly loaded onto biomaterial scaffolds, which are then implanted into the uterine cavity or infused intravenously. These treatments can regulate various signaling pathways and participate in processes such as proliferation, apoptosis, migration, invasion, differentiation, fibrosis, immunomodulation, inflammatory responses, and EMT of stromal cells and endometrial epithelial cells, affecting immune cells and vascular endothelial cells, thereby promoting endometrial injury repair and improving reproductive outcomes (Table 2).

Table 2 A summary of the literature on human umbilical cord mesenchymal stem cells and their exosomes in endometrial injury repair.

No.	MSC origin	Vehicle type	Carrier	Stem cell transplant method	Model	Target (gene/pathway)	Biological functions	Reproductive outcome	Ref.
1	HucMSC	NA	HA/Gel hydrogel	Intrauterine injection	Endometrial mechanical injury rat	MEK/ERK1/2	Fibrosis; inflammation; migration; proliferation	Endometrial thickness; number of glands; endometrial vessels; embryo implantation rate; live birth rate	[30]
2	HucMSC	NA	NA	NA	HESC	CCL2, HGF	Migration; invasion	NA	[90]
3	HucMSC	Exosome	NA	Femoral vein injection	Endometrial 95% ethanol injury rat	MiR-202-3p/MMP11	ECM remodeling	NA	[65]
4	HucMSC	NA	GelMA/SerMA	Intrauterine injection	Endometrial 95% ethanol injury mouse	CD31, CK18, Ki67, vimentin, caspase 3	Angiogenesis; proliferation; apoptosis; fibrosis	Endometrial thickness; embryo implantation rate	[58]
5	T10-HucMSC	Exosome	NA	Intrauterine injection	HESC treated with TGF-β endometrial mechanical injury rat	MiR-543N-cadherin α-SMA FN1	Fibrosis; EMT; differentiation; apoptosis; migration	NA	[59]
6	HucMSC treated with TGF-β1	Exosome	Cross-linked sodium hyaluronate	Intrauterine injection	Endometrial 95% ethanol injury rat	TGF-β1/Smad2/3	Angiogenesis; fibrosis; proliferation; migration; collagen remodeling	Endometrial thickness; number of glands; embryo implantation rate	[60]
7	HucMSC	Exosome	NA	NA	EEC injury induced by hypoxia	MiR-663a/CDKN2A	Proliferation; apoptosis; migration; EMT	NA	[61]
8	T10-HucMSC	Exosome	NA	Intrauterine injection	Endometrial 95% ethanol injury mouse	HOXA10	Fibrosis; angiogenesis; apoptosis	Endometrial thickness; number of glands; embryo implantation rate; Endometrial receptivity	[62]
9	UC-MSCbFGF	NA	Collagen scaffolds	Intrauterine injection	Uterine full-thickness defect rat	NA	Inflammation; angiogenesis; proliferation; regeneration	Endometrial thickness; embryo implantation rate	[91]
10	HucMSC	NA	PF-127	Intrauterine injection	Endometrial 95% ethanol injury rat	IL-1β, bFGF, EGF, HGF	Angiogenesis; inflammation; fibrosis	Endometrial thickness; number of glands	[65]
11	HucMSC	NA	SF-SIS scaffolds	Transplanted in situ	Uterine full-thickness defect mouse	circPTP4A2/miR-330-5p/PDK2	Fibrosis; mitochondrial metabolism	Number of glands; endometrial fibrosis area	[66]
12	HucMSC	NA	NA	Tail vein injection versus intrauterine injection	Endometrial 95% ethanol injury rat	IL-1β, TNF-α, bFGF	Angiogenesis; inflammation	Number of glands; endometrial receptivity	[92]
13	HucMSC	Exosome	NA	Tail vein injection	Mifepristone-induced HESC injury endometrial 95% ethanol injury rat	Integrin alpha, thrombospondin, laminin, collagen, VWF	ECM remodeling; protein degradation and absorption; inflammation; proliferation; apoptosis; migration; adhesion	Endometrial morphology and thickness; number of glands; endometrial fibrosis area; embryo implantation rate	[68]
14	HucMSC	NA	NA	NA	Endometrial mechanical injury mouse	MiR-455-5p/SOCS3/JAK/STAT455	Fibrosis; proliferation; cell cycle progression	Number of glands; endometrial fibrosis area	[69]
15	HucMSC	NA	AAM	Transplanted in situ	Endometrial 95% ethanol injury rat	Keratin, vimentin, integrin β3, VEGF, MMP9	ECM remodeling; proliferation; migration; inflammation	Endometrial morphology and thickness; number of glands; endometrial receptivity; pregnancy rate; birth weight; number of neonatal rats	[70]
16	HucMSC	NA	Collagen scaffolds	Hysteroscopic transplantation	16 females experiencing infertility with thin endometrium (embryo transfers ≤ 5.5 mm)	ERα, PR	Angiogenesis; proliferation; hormone response; differentiation; angiogenesis	Endometrial thickness; uterine receptivity (endometrial volume and subendometrial blood flow); number of glands; miscarriage rate; pregnancy rate; live birth rate; birth defects	[55]
17	HucMSC	NA	NA	Intraperitoneal injection	Endometrial mechanical injury rat	TGF-β1/Smad3	Fibrosis; angiogenesis; differentiation; migration	Endometrial thickness; endometrial fibrosis area; number of glands; pregnancy rate; number of fetuses	[93]
18	HucMSC	Exosome	Collagen scaffolds	Transplanted in situ	Endometrial mechanical injury rat	MiR-223-3p	Immunomodulation (infiltration and polarization of macrophages); collagen remodeling; hormone response; inflammation; endometrial regeneration; fibrosis; angiogenesis	Pregnancy rate	[71]
19	HucMSC	NA	HA-GEL	Transplanted in situ	Endometrial mechanical injury rhesus monkey	IL-4, IGF-1, EGF, IFN, VEGF	Fibrosis; angiogenesis; inflammation	Endometrial morphology and thickness; number of glands; endometrial receptivity	[84]
20	HucMSC	NA	Collagen scaffolds	Intrauterine injection	Endometrial mechanical injury rat	ER, CTGF, TGFβ1, FGF2	Proliferation; hormone response; fibrosis	Number of glands; endometrial fibrosis area	[94]
21	HucMSC	Exosome	NA	NA	OGD/R-induced injured EEC	TLR4/RelA	Cell viability; cell death; inflammation	NA	[95]
22	HucMSC	NA	Collagen scaffolds	Transplanted in situ	HESC endometrial mechanical injury rat	TGF-b1, PDGF-BB, VEGF-A, Ki67	Proliferation; apoptosis; hormone response; regeneration; collagen remodeling; fibrosis; angiogenesis	Endometrial morphology and thickness; number of glands; pregnancy rate	[96]
23	HucMSC	NA	Collagen scaffolds	Transplanted in situ (spread onto an 18F Foley catheter and placed into the uterine cavity)	26 females experiencing infertility with recurrent IUA	ERα, vimentin, Ki67, VWF, ΔNP63	Proliferation; differentiation; angiogenesis	IUA score; endometrial thickness; ongoing pregnancy rate (> 12 weeks active fetus); live birth rate; menstrual volume; miscarriage rate; histological changes in endometrium	[97]
24	HucMSC	NA	NA	Tail vein injection	Endometrial 95% ethanol injury rat	VEGF-A, MMP9, IFN-γ, TNF-α, IL-2, α-SMA, TGF-β	Fibrosis; proliferation; angiogenesis; inflammation; differentiation; regeneration; fibrosis	Endometrial morphology and thickness; number of glands; endometrial fibrosis area; pregnancy rate	[98]
25	HucMSC	NA	Collagen scaffolds	Intrauterine injection	Uterine full-thickness defect rat	MMP-9, α-SMA, VWF	Adhesion; migration; differentiation; angiogenesis; differentiation	Endometrial morphology and thickness; number of glands; pregnancy rate; number, size, and weight of fetuses	[56]
26	HucMSC	Exosome	NA	NA	Mifepristone-induced HESC injury	PTEN/AKT	Proliferation; apoptosis	NA	[57]
27	HucMSC	Exosome	NA	NA	Mifepristone-induced HESC injury	MiR-7162-3p/APOL6	Apoptosis; regeneration	NA	[58]
28	HucMSC	Exosome	NA	NA	HESC treated with TGF-β1	MiR-145-5p/ZEB2	Fibrosis	NA	[54]
29	HucMSC	Exosome	NA	NA	HESC	NA	Proliferation	NA	[99]
30	WJMSC	NA	NA	Intrauterine injection	1 female experiencing infertility with IUA; 1 female experiencing infertility with endometrial atrophy	NA	NA	Endometrial thickness; pregnancy rate	[80]
31	WJMSC	NA	NA	NA	Mifepristone-induced HESC injury	Circ6401miR-29b-1-5p/RAP1B	Angiogenesis; apoptosis	NA	[100]
32	WJMSC	NA	NA	Intrauterine injection	Endometrial 95% ethanol injury rat	TGF-β1 Rho/ROCK	Angiogenesis; regeneration	Endometrial thickness; number of glands	[101]
33	hAMSC	NA	NA	NA	Mifepristone-induced HESC injury	VEGF, caspase 3, caspase 8	Proliferation; apoptosis	NA	[102]
34	hAMSC	NA	NA	Intrauterine injection	Endometrial 95% ethanol injury rat	Notch pathway	Differentiation; regeneration and repair	NA	[103]
35	hAMSC	NA	NA	Intrauterine injection	Endometrial mechanical injury rat	TNFα, IL-1β, bFGF, IL-6, VEGF	Angiogenesis; regeneration	Endometrial thickness; number of glands; endometrial fibrosis area	[52]

AKT: Protein kinase B; APOL6: Apolipoprotein L6; bFGF: Basic fibroblast growth factor; CCL2: C-C motif ligand 2; CTGF: Connective tissue growth factor; ECM: Extracellular matrix; EEC: Endometrial epithelial cell; EGF: Epidermal growth factor; EMT: Epithelial-mesenchymal transition; ER: Estrogen receptor; ERα: Estrogen receptor α; ERK1/2: Extracellular regulated protein kinases 1/2; FGF2: Fibroblast growth factor 2; hAMSC: Human amniotic mesenchymal stromal cell; HA: Hyaluronic acid; HESC: Human embryonic stem cell; HGF: Hepatocyte growth factor; HOXA10: Homeobox A10; HucMSC: Human umbilical cord mesenchymal stem cell; IFN: Interferon; IGF-1: Insulin-like growth factor-1; IL: Interleukin; IUA: Intrauterine adhesions; JAK: Janus kinase; MEK: Mitogen-activated and extracellular signal-regulated kinase; miR: MicroRNA; MMP: Matrix metalloproteinase; MSC: Mesenchymal stem cell; NA: Not available; PDGF-BB: Platelet-derived growth factor-BB; PR: Progesterone receptor; PTEN: Phosphatase and tensin homolog; SF-SIS: Silk fibroin small-intestinal submucosa; α-SMA: α-smooth muscle actin; Smad: Small mothers against decapentaplegic; STAT455: Signal transducer and activator of transcription 455; TGF-β: Transforming growth factor-β; TLR4: Toll-like receptor 4; TNF: Tumor necrosis factor; UC-MSC^bFGF: Umbilical cord-mesenchymal stem cells^{basic fibroblast growth factor}; VEGF: Vascular endothelial growth factor; VWF: Von Willebrand factor; WJMSC: Wharton jelly of umbilical cord; ZEB2: Zinc finger E-box binding homeobox 2 axis.

HucMSC therapy operates through three pathways: (1) Transdifferentiation; (2) Paracrine effect; and (3) Immunomodulation. Overall, hucMSCs migrate and localize to the injured site, differentiating into functional cells. Additionally, hucMSCs release growth factors and EVs through paracrine secretion, playing roles not only in cell proliferation, apoptosis, fibrosis, EMT, and angiogenesis but also in regulating the immune system to enhance tissue regeneration and control inflammation (Figure 2).

Figure 2 Mechanisms of mesenchymal stem cell-mediated repair in endometrial injury. This figure illustrates how human umbilical cord mesenchymal stem cells aid in endometrial repair through transdifferentiation, paracrine effects [via microRNAs (miRNA), exosomes, and growth factors], and immunomodulation [involving interactions with T cells, B cells, natural killer (NK) cells, and macrophages].

When the endometrial basal layer is injured (such as during artificial abortion, infection, inflammation, and uterine artery embolization), isolated and purified MSCs from several tissues, such as adipose tissues, umbilical cord, placenta, menstrual blood, and bone marrow, contribute to endometrial repair through transdifferentiating, paracrine, and immunomodulatory actions.

Regulation of cell proliferation and apoptosis: Human endometrium is a highly dynamic and regenerative tissue, and cell apoptosis and proliferation are crucial for endometrial regeneration and repair. A study conducted in 2014 found that treatment of IUA mice with bone marrow MSCs resulted in the detection of only a small amount of exogenous MSCs in the endometrium of the transplanted mice[52]. Furthermore, in rodent animal models, non-hematopoietic stem cell implantation did not lead to the formation of endometrial cell lines[53]. Therefore, it is suggested that, besides directly differentiating into endometrial cells, MSCs may also promote endometrial injury repair by regulating endometrial cell proliferation and apoptosis through paracrine actions.

Lv et al[54] demonstrated that hucMSC-EVs promote endometrial proliferation in a dose-dependent manner. Among 16 females experiencing infertility with thin endometrium, implantation of hucMSC-EVs loaded on collagen scaffolds led to increased endometrial cell proliferation, substantial endometrial thickening, and increased gland numbers[55]. HucMSC-EVs also inhibit the apoptosis of damaged endometrial stromal cells (human endometrial stromal cells) induced by mifepristone through miR-7162-3p/apolipoprotein L6 and phosphatase and tensin homolog/protein kinase B axis, thus promoting endometrial regeneration[56,57].

Anti-fibrosis activity: Normal endometrium undergoes natural repair and regeneration during the menstrual cycle, without scarring or functional loss. However, damaged endometrium cannot be normally repaired, becoming prone to fibrosis, scarring, and adhesions. This can lead to infertility caused by embryo implantation failure. Therefore, antifibrosis is essential for endometrial regeneration and repair. Various factors promoting and degrading organ fibrosis have been found to be inseparably linked to endometrial injury, such as basic fibroblast growth factor, TGF-β, Smad3, a disintegrin and metalloproteinase-15, platelet-derived growth factor, and a disintegrin and metalloproteinase-17. Among them, TGF-β mediates extracellular matrix (ECM) deposition, induces EMT, and facilitates epithelial cell differentiation into myofibroblasts, making it the strongest promoter of fibrosis.

By mediating the miR-145-5p/zinc finger E-box binding homeobox 2 axis, hucMSC-EVs inhibit endometrial fibrosis induced by TGF-β1[58]. Injecting methacrylate sericin and methacrylate gelatin hydrogels encapsulating hucMSCs into the uterine cavity leads to a remarkable decline in fibrotic endometrial area and collagen content, along with an elevation in endometrial thickness and embryo implantation rate in mice[59]. Endometrial fibrosis is prevented by hucMSC-EVs carrying miR-543 through N-cadherin downregulation, both in vivo and in vitro[60]. Additionally, the fibrotic endometrial area in IUA mice is notably decreased by hucMSCs encapsulated in silk fibroin small-intestinal submucosa[61]. The miR-455-5p upregulation in hucMSCs inhibits endometrial fibrosis in mice[62]. These findings indicate that hucMSCs or hucMSC-EVs can modulate endometrial fibrosis through multiple signaling pathways, thus improving endometrial receptivity.

Induction of EMT: EMT refers to a process in which epithelial cell cytoskeleton, under the influence of changes in the surrounding microenvironment (such as inflammation and injury), loses its native shape. Then, the cells become spindle-shaped, with a loss of polarity and dissolution of intercellular junctions. As a result, tightly connected epithelial cells transform into loose, low-differentiated, and highly proliferative stromal cells. EMT occurs in three stages: (1) Transformation of epithelial cells into interstitial direction during embryonic development; (2) Conversion of epithelial cells into fibroblasts during inflammatory responses, promoting the formation of fibrosis; and (3) Loss of polarity and surface adhesion molecules by epithelial cells during the invasion and metastasis of epithelial tumors, resulting in their transformation into the interstitial space. Under normal physiological conditions, the regeneration of uterine stroma and epithelial cells is promoted by the differentiation of endometrial stromal cells and their integration into the endometrial epithelium, highlighting the critical role of EMT in endometrial regeneration.

Emerging studies have suggested that hypoxia-induced damage in endometrial adenocarcinoma cells may involve modulation of key signaling pathways associated with EMT[63]. For example, Yuan et al[60] demonstrated that hucMSC-EVs carrying miR-543 regulate TGF-β1-triggered EMT in IUA mouse models and human endometrial epithelial cells. However, further investigations are required to clarify the exact mechanisms and therapeutic potential.

Reconstruction of ECM: As a highly dynamic structural network, the ECM undergoes continuous remodeling mediated by various matrix degradation enzymes, providing both mechanical and structural support for cells and tissues. It not only regulates signaling space transmission by binding soluble ligands and transmembrane receptors, but also modulates cell proliferation, migration, and differentiation in response to chemical and mechanical signals to maintain tissue function and homeostasis. However, when the endometrium is injured, the fibroblasts in the damaged area are activated through multiple signaling pathways, differentiate into myofibroblasts, and secrete ECM components to restore ECM structure. This repair process is disordered, resulting in the replacement of functional tissue with a stiff and disordered ECM[64], which is strongly correlated with fibrosis.

MiR-202-3p overexpression in hucMSC-EVs can promote endometrial repair by ECM remodeling through regulating target gene expression associated with cell differentiation, migration, and proliferation[65]. Moreover, ECM degradation is the main cause of endometrial injury, and ECM reconstruction occurs during the transplantation and repair process of hucMSCs, as revealed by Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses[66]. HucMSCs combined with human acellular amniotic matrix contribute to endometrial repair through ECM remodeling[67].

Promotion of angiogenesis: Endometrial injury results in an increase in uterine artery resistance, the suppression of new vessel formation, and the occurrence of ischemia and hypoxia, leading to loss of nutrition support for the endometrium and impairing endometrial regeneration. Therefore, angiogenesis is essential in endometrial regeneration and repair.

HucMSC-EVs can increase endometrial microvascular density in rats with thin endometrium through the TGF-β1/Smad2/3 signaling pathway, thereby improving pregnancy rates[68]. Overexpression of homeobox A10 in hucMSC-EVs reduces uterine blood flow resistance and vascular endothelial growth factor (VEGF) expression in mice with injured endometrium, promoting endometrial angiogenesis and improving reproductive outcomes[69]. In rats with thin endometrium treated with pluronic F-127s/hucMSCs, the abundance of vascular markers, such as nitric oxide synthase 3 and VEGFA, was raised, along with a marked elevation in the number of von Willebrand factor and VEGFA-positive vessels. Such results indicate that hucMSCs promote the angiogenic factor release, thereby enhancing the regeneration of thin endometrium[70]. In 2018, significant improvement in uterine blood flow was observed in 26 Chinese females experiencing infertility with thin endometrium who received intramuscular injections of hucMSC-EVs[22]. By integrating hucMSC-EVs into collagen scaffolds and implanting them into rats with endometrial injury, miRNA-223-3 significantly promoted angiogenesis and improved pregnancy rates[71].

Participation in immunomodulation: MSC-mediated immunomodulation depends on direct cell-cell interactions or cytokine secretion. Immunoregulation is achieved through interactions with monocytes/macrophages, T cells, dendritic cells, natural killer (NK) cells, and B cells. Notably, anti-inflammatory monocytes/macrophages and regulatory T cells (Treg) play crucial roles in immunomodulation[72]. MSCs and their EVs have been confirmed to participate in immunomodulation, including: (1) Regulating the state of T cells, such as suppressing T cell activation, facilitating T cell apoptosis, and promoting Treg cell generation[73]; (2) Enhancing M2 macrophage polarization and suppressing M1 macrophage polarization[74]; (3) Inhibiting B cell differentiation and modulating B cell induction[75]; (4) Recruiting NK cells to migrate to inflammatory sites and exert immune effects and inhibiting NK cell proliferation and cytokine secretion; and (5) Suppressing endocytosis and secretion of interleukin (IL)-12 to inhibit dendritic cell activation[76]. Therefore, MSCs possess strong immunoregulatory capabilities, regulating immune cell activity and controlling inflammatory responses through their surface molecules, cytokine secretion, and other cell functions.

The immune mechanism underlying endometrial injury remains unclear and may be related to vaginal microbial ecological imbalance (caused by frequent sexual intercourse, long-term antibiotic use, and decreased estrogen) and damage to the endometrial surface barrier (due to repeated manipulation of endometrial cavity). In 2022, Hua et al[77] found that in the IUA rabbit model, hucMSCs could differentiate into macrophages to participate in immunomodulation, secrete immunoregulatory factors, such as IL-6, IL-8, monocyte chemoattractant protein-1, CC-motif chemokine ligand 5, and toll-like receptor 4, to inhibit inflammation, and regulate the T helper 17/Treg balance through the nuclear factor-κB signaling pathway to suppress the immune response to injured endometrium. However, the specific mechanism involved requires further investigation with larger-scale basic and clinical studies.

Repair of damaged endometrium by miRNA: As a non-coding RNA that contains roughly 22 nucleotides, miRNA modulates gene expression through direct binding to the 3’ untranslated region of its target genes. As demonstrated by Azizi et al[78], miRNA is strongly related to scar repair, participating in regulating angiogenesis, inflammatory responses, collagen deposition, cell apoptosis and proliferation, and the expression and function of ECM components. Currently, more than 1000 miRNAs are encoded in the human genome, among which 56 exhibit significant differences in expression between normal endometrium and IUA tissues[79]. In recent years, EV-derived miRNAs have received considerable attention due to their resistance to RNAse degradation and stability in circulation. As effective carriers, these miRNAs can deliver miRNA to target cells to exert specific regulatory effects.

Multiple miRNAs have been confirmed to be involved in hucMSC-mediated repair of damaged endometrium. HucMSC-EVs have been reported to mediate miR-663a/cyclin dependent kinase inhibitor 2A to regulate the apoptosis, EMT, and proliferation of hypoxia-damaged endometrial adenocarcinoma cells and repair endometrial injury[63]. Wang et al[65] discovered that hucMSC-EVs promoted the repair of damaged endometrium through miR-202-3p upregulating collagen type I alpha 1 chain, collagen type III alpha 1 chain, collagen VI, and fibronectin. MiR-543 overexpression in hucMSC-EVs downregulates N-cadherin, preventing endometrial fibrosis[60]. HucMSCs regulate circular RNA protein tyrosine phosphatase 4A2/miR-330-5p/pyruvate dehydrogenase kinase 2 to improve IUA injury in mice, reducing endometrial fibrosis and fibrosis area and increase the endometrial gland numbers[61]. Additionally, miR-455-5p[62], miR-7162-3p[57], miR-223-3p[71], miR-145-5p[58], and miR-29b-1-5[80] have been reported to be associated with hucMSCs or hucMSC-EVs in promoting endometrial repair.

Loading of stem cells onto scaffolds/hydrogels

The transplantation methods for hucMSCs and hucMSC-EVs in treating endometrial injury include intrauterine infusion, intravenous injection, intraperitoneal injection, and stem cell-loaded scaffolds/hydrogels. However, during intrauterine infusion, there is an increased risk of stem cell suspension loss, resulting in reduced retention and survival rates of stem cells. During intravenous and intraperitoneal injections, stem cell migration and homing to the endometrium are limited, lowering their utilization rate. In contrast, scaffolds/hydrogels are capable of providing mechanical and structural support for stem cell survival, migration, and development, extending stem cell preservation time and facilitating endometrial regeneration, such as the promotion of angiogenesis the reduction in inflammatory responses, anti-fibrosis, and immunomodulation[81]. Therefore, some scholars have proposed that stem cells and scaffolds are two core components of endometrial repair, both of which have become hot topics in regenerative medicine[82,83].

The application of collagen scaffolds is the most widespread. Numerous studies have demonstrated that loading hucMSCs and hucMSC-EVs onto collagen scaffolds can significantly improve the fertility outcomes in rats with endometrial injury and in females experiencing infertility with thin endometrium, even achieving live births. In 2023, Zhang et al[30] loaded hucMSCs onto a hydrazide-grafted gelatin and oxidized hyaluronic acid (HA)-based HA/Gel hydrogels, which were then injected into the uterine cavity of rats with endometrial injury. Their findings revealed thickened endometrium, an increase in gland and vessel numbers, along with a marked improvement in live birth and embryo implantation rates.

Chen et al[59] encapsulated hucMSCs in methacrylate gelatin/methacrylate sericin hydrogels and facilitated fertility recovery and endometrial regeneration in mice through in situ injection. Zheng et al[61] constructed a scaffold seeded with hucMSCs and loaded it onto silk fibroin small-intestinal submucosa scaffolds, which repaired damaged endometrium in IUA mice. In a rhesus monkey model with endometrial injury, hucMSC-loaded auto-crosslinked HA gel exhibited anti-adhesion properties and promoted endometrial regeneration[84]. Therefore, scaffolds that simultaneously possess mechanical support and promote regenerative biological effects have emerged as an essential part of stem cells and their EV in treating endometrial injury.

PERSPECTIVES

While hucMSCs and hucMSC-EVs show considerable promise for endometrial repair, several challenges and uncertainties must be addressed before clinical application. The key issues include: (1) No international unified standard exists for the isolation, culture, identification, preparation, and administration of hucMSCs and hucMSC-EVs, which hinders reproducibility and scalability; (2) The parameters, such as the appropriate number of transplanted cells, timing, frequency, treatment cycles, and administration routes, have not been established, which limits the development of effective protocols; (3) MSC-based treatment may have potential risks, such as alterations in immune function and possible tumorigenicity. Although no cases of MSC-related malignancy have been reported, large-scale, long-term prospective studies are required to confirm their safety; and (4) The exact mechanism by which damaged endometrium signals MSCs to initiate the repair mechanism remains unclear. A deeper understanding of these mechanisms is essential for optimizing MSC therapy. Addressing these gaps will be crucial to optimizing MSC therapy for endometrial injury repair.

CONCLUSION

In summary, hucMSCs and hucMSC-EVs offer a promising regenerative approach for treating endometrial injury by modulating key processes, such as cell apoptosis, proliferation, fibrosis, EMT, ECM remodeling, angiogenesis, and immunomodulation. These mechanisms collectively enhance endometrial receptivity and improve reproductive outcomes. As further research addresses existing challenges and standardizes protocols, hucMSC-based therapies hold potential as a viable and safe option for females experiencing infertility with endometrial dysfunction, offering new hope for improving reproductive health and advancing the field of regenerative medicine.