Research Progress of Stem Cell Therapy for Premature Ovarian Insufficiency

Abstract

Premature ovarian insufficiency (POI) is a heterogeneous reproductive and endocrine disorder characterized by impaired ovarian function before the age of 40 years. Conventional management, particularly hormone therapy, addresses hypoestrogenic symptoms and long-term health risks but does not reliably restore endogenous ovarian activity or fertility. Stem cell-based strategies have therefore attracted considerable interest because of their potential paracrine, immunomodulatory, anti-apoptotic, angiogenic, and microenvironment-remodeling effects. This review summarizes current evidence on stem cell sources, therapeutic mechanisms, preclinical models, clinical studies, extracellular vesicle-based approaches, biomaterials, gene-editing strategies, and translational challenges in POI. Particular emphasis is placed on the distinction between mechanistic and animal evidence, uncontrolled or pilot human studies, and controlled clinical evidence. Across preclinical models, mesenchymal stem cells (MSCs) frequently improve ovarian histology, hormone profiles, follicular parameters, and reproductive performance; however, these findings cannot be assumed to represent restoration of ovarian reserve or fertility in humans. Clinical evidence remains limited, heterogeneous, and predominantly derived from small non-randomized studies, with outcomes ranging from menstrual recovery and changes in AMH, FSH, estradiol, or antral follicle count to a small number of reported pregnancies. These surrogate changes should be distinguished from clinically meaningful endpoints such as ovulation, ongoing pregnancy, and live birth. Emerging approaches involving extracellular vesicles, biomaterials, and gene editing may improve cell retention or therapeutic potency, but most remain preclinical or early translational strategies. Overall, stem cell therapy should currently be regarded as an investigational approach for POI. Progress toward clinical translation will require standardized cell products, clearly defined dosing and administration routes, appropriate comparator groups, clinically meaningful reproductive endpoints, rigorous long-term safety surveillance, and adequately powered controlled trials.

Introduction

Premature ovarian insufficiency (POI), also known as premature ovarian insufficiency (POI), is a significant reproductive disorder affecting approximately 1% to 3% of women under the age of 40, with an increasing incidence worldwide¹ Characterized by amenorrhea, hypoestrogenism, elevated gonadotropin levels, and infertility, POI leads to the premature depletion or dysfunction of ovarian follicles, resulting in diminished ovarian reserve and impaired reproductive capacity² The etiology of POI is multifactorial, encompassing genetic abnormalities, autoimmune disorders, iatrogenic factors such as chemotherapy and radiotherapy, oxidative stress, infections, and environmental influences. These diverse pathogenic mechanisms culminate in follicular atresia, granulosa cell apoptosis, and disruption of ovarian microenvironment, ultimately causing ovarian failure² The clinical manifestations of POI extend beyond infertility, including menopausal symptoms, increased risk of osteoporosis, cardiovascular disease, psychological distress, and reduced quality of life. Given the profound impact of POI on women’s health, effective therapeutic strategies are urgently needed.

Currently, hormone replacement therapy (HRT) remains the mainstay treatment for POI, aiming to alleviate hypoestrogenic symptoms and reduce associated health risks³ However, HRT does not restore ovarian function or fertility, and its long-term use is associated with increased risks of thromboembolism, breast cancer, and cardiovascular events, limiting its applicability. Assisted reproductive technologies (ART), including oocyte donation, provide options for achieving pregnancy but are not suitable for all patients and do not address the underlying ovarian insufficiency⁴ Moreover, fertility preservation methods such as ovarian tissue cryopreservation and transplantation have been developed, particularly for cancer patients undergoing gonadotoxic treatments, but these approaches have limitations related to tissue viability, ischemia, and risk of reintroducing malignant cells. Consequently, there is a critical need for novel therapies that can restore ovarian function and fertility in POI patients.⁵

In recent years, stem cell-based therapies have emerged as promising regenerative approaches for POI, leveraging the self-renewal, differentiation potential, and paracrine effects of stem cells to repair damaged ovarian tissue.^6–8 Among various stem cell types, mesenchymal stem cells (MSCs) have attracted considerable attention due to their low immunogenicity, ease of isolation from multiple tissue sources (bone marrow, umbilical cord, adipose tissue, menstrual blood), and potent immunomodulatory and trophic properties.⁹ Preclinical studies in animal models of chemotherapy-induced POI have demonstrated that MSC transplantation can restore ovarian morphology, increase follicle numbers, normalize hormone levels, and improve fertility outcomes. The mechanisms underlying these therapeutic effects include homing of MSCs to injured ovaries, differentiation into granulosa-like cells, secretion of growth factors promoting angiogenesis and follicular development, inhibition of granulosa cell apoptosis, and modulation of the ovarian immune microenvironment.^10–12 Additionally, MSC-derived extracellular vesicles and exosomes have shown potential as cell-free therapies, delivering bioactive molecules that mitigate oxidative stress, inflammation, and cell death in ovarian tissue.^13–15

Despite encouraging preclinical data, clinical translation of stem cell therapies for POI faces challenges including heterogeneity of MSC populations, optimal cell source and dosage, delivery methods, safety concerns, and long-term efficacy.¹⁶ Recent advances in engineering MSCs, such as selecting specific subpopulations with enhanced regenerative capacity or modifying epigenetic profiles, aim to overcome these limitations and improve therapeutic outcomes.¹⁷ Furthermore, ongoing clinical trials are evaluating the safety and efficacy of MSC transplantation in POI patients, with preliminary results indicating restoration of ovarian function and occasional pregnancies. However, larger, well-controlled studies are needed to establish standardized protocols and confirm long-term benefits.¹⁸

In addition to MSCs, other stem cell types including human pluripotent stem cells (hPSCs), embryonic stem cells (hESCs), and induced pluripotent stem cells (hiPSCs) have been investigated for their ability to differentiate into oocyte-like cells and granulosa cells, offering potential avenues for in vitro folliculogenesis and fertility restoration.¹⁹ These approaches may complement MSC-based therapies by providing a source of functional germ cells, although ethical and technical challenges remain. Moreover, strategies such as in vitro activation (IVA) of dormant follicles and mitochondrial replacement therapy are being explored to enhance ovarian reserve and oocyte quality in POI patients.^20,21

Given the heterogeneity of POI and the rapid expansion of regenerative strategies, interpretation of stem cell research requires a clear distinction between biological activity, improvement in ovarian function, preservation or enhancement of ovarian reserve, and demonstrated reproductive benefit.²² Recent reviews and systematic analyses have summarized MSC-based therapy, extracellular vesicles, tissue engineering, and transplantation strategies, but differ in scope and emphasis.^23,24 The present review complements these reports by organizing the evidence along a translational continuum and comparing stem-cell sources, mechanisms, administration routes, efficacy endpoints, safety considerations, and translational readiness.²⁵ Particular attention is given to the strength of evidence supporting each outcome and to the distinction between surrogate ovarian markers and clinically meaningful fertility outcomes.

Literature Search and Review Methodology

This review used a structured literature-search and narrative-synthesis approach. The reporting principles of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 statement were used to improve transparency of study identification and selection.²⁶ PubMed/MEDLINE, Embase, Web of Science Core Collection, Scopus, and Google Scholar were searched from database inception to August 2026. Reference lists of relevant reviews and primary studies were also screened. Core search concepts combined terms related to “premature ovarian insufficiency”, “premature ovarian insufficiency”, “stem cells”, “mesenchymal stem cells”, “bone marrow”, “umbilical cord”, “adipose-derived stem cells”, “extracellular vesicles”, “exosomes”, “ovarian regeneration”, and “fertility”.

Studies were considered when they investigated stem-cell-based or stem-cell-derived interventions relevant to POI and reported mechanistic, histological, endocrine, follicular, reproductive, safety, or clinical outcomes. In vitro studies, animal studies, observational or uncontrolled human studies, controlled clinical studies, and relevant systematic reviews or meta-analyses were considered according to their evidentiary role. Editorials, commentaries, duplicate publications, and studies unrelated to ovarian insufficiency were excluded. Clinical trials identified only through trial registries were treated as registered studies rather than evidence of efficacy unless peer-reviewed outcome data were available.

The evidence was synthesized according to four broad levels: (1) in vitro and mechanistic evidence; (2) animal studies; (3) uncontrolled, pilot, or case-based human studies; and (4) controlled clinical studies. Study interpretation considered design, sample size, comparator availability, cell source, cell dose, administration route, follow-up duration, and outcome type. Greater interpretive weight was assigned to controlled prospective studies and clinically meaningful reproductive outcomes than to uncontrolled changes in surrogate biomarkers. Because of substantial heterogeneity in animal models, cell products, dosing, administration routes, patient populations, outcome definitions, and follow-up, a quantitative meta-analysis was not attempted. Methodological limitations and translational readiness are therefore emphasized throughout the review (Table 1 and Figure 1).

Table 1.

Evidence Hierarchy and Translational Value of Stem Cell Therapy for Premature Ovarian Insufficiency

Evidence Level	Typical Evidence	Primary Value	Key Limitation
Mechanistic	In vitro/pathway studies	Biological plausibility	Cannot establish tissue or clinical efficacy
Preclinical	Animal POI models	Histology, hormones, follicles, reproductive performance	Model heterogeneity and limited human representativeness
Early human	Case reports/series; pilot or non-randomized studies	Feasibility, safety signals, biological activity	Small samples, no/limited controls and confounding
Controlled clinical	Controlled prospective or randomized studies	Comparative efficacy and safety	Currently limited number, sample size, endpoints and follow-up

Figure 1.

Evidence-to-translation framework for stem cell therapy in POI. The framework distinguishes mechanistic, preclinical, early human, and controlled clinical evidence and emphasizes the hierarchy from surrogate ovarian markers to clinically meaningful reproductive outcomes.

Pathophysiology and Current Treatment of Primary Ovarian Insufficiency

Definition and Epidemiology of Premature Ovarian Insufficiency (POI)

Premature ovarian insufficiency (POI) is the preferred contemporary term for impaired or lost ovarian function before 40 years of age.¹⁶ Unlike the term “premature ovarian failure”, POI reflects the heterogeneous and potentially intermittent nature of ovarian activity, including occasional follicular activity or spontaneous ovulation.¹⁷ The 2024 international guideline recommends diagnosing POI in women younger than 40 years on the basis of disordered menstrual cycles together with biochemical evidence of ovarian insufficiency; a fixed FSH threshold such as >40 IU/L should not be presented as an obligatory diagnostic criterion. FSH should be interpreted in the clinical context, with alternative causes of menstrual disturbance excluded.²⁷

POI affects approximately 1% of women globally, although estimates vary according to population characteristics and diagnostic criteria. Etiologies include genetic abnormalities, autoimmune disease, iatrogenic injury from chemotherapy or radiotherapy, and idiopathic cases.²⁸ The consequences extend beyond infertility and hypoestrogenic symptoms to bone, cardiovascular, sexual, psychological, and neurological health. Current international guidance emphasizes individualized evaluation of etiology, comorbidities, fertility goals, and long-term health risks.²⁷

For the purposes of this review, ovarian function, ovarian reserve, and fertility are treated as related but non-equivalent concepts. Changes in FSH, estradiol, AMH, antral follicle count (AFC), or ovarian volume may indicate altered endocrine activity or follicular dynamics, but none alone demonstrates restoration of fertility. Accordingly, “ovarian function improvement” refers to endocrine or follicular changes, whereas reproductive efficacy refers to ovulation, pregnancy, ongoing pregnancy, and live birth.

Causes and Pathogenesis of Premature Ovarian Insufficiency

Premature ovarian insufficiency (POI), also known as premature ovarian insufficiency (POI), is a multifactorial disorder characterized by the loss of ovarian function before the age of 40, leading to amenorrhea, hypergonadotropic hypogonadism, estrogen deficiency, and infertility. The etiology and pathogenesis of POI are complex and involve genetic, autoimmune, iatrogenic, environmental, and infectious factors, each contributing to follicular depletion or dysfunction through distinct mechanisms.

Genetic factors play a significant role in the development of POI. Chromosomal abnormalities, particularly involving the X chromosome, are well-documented causes. Turner syndrome mosaicism, characterized by partial or complete monosomy of the X chromosome, leads to accelerated follicular atresia and ovarian dysgenesis. In addition to gross chromosomal anomalies, mutations in autosomal genes critical for ovarian development and function have been implicated. For instance, premutation expansions in the FMR1 gene are associated with fragile X-associated premature ovarian insufficiency, while mutations in BMP15 and GDF9, both members of the transforming growth factor-beta superfamily, disrupt folliculogenesis by impairing granulosa cell function and oocyte maturation. These genetic defects often result in diminished ovarian reserve and premature follicle loss, underscoring the importance of genetic screening in POI diagnosis and management.²⁹

Autoimmune mechanisms contribute to approximately 4% to 30% of POI cases, where the immune system aberrantly targets ovarian tissue. Autoimmune POI is frequently associated with other autoimmune disorders such as Addison’s disease and autoimmune thyroiditis. The presence of anti-ovarian antibodies suggests an immunologic attack on ovarian follicles, leading to their destruction and subsequent ovarian failure. Experimental models and clinical studies have demonstrated that mesenchymal stem cells (MSCs) can modulate immune responses, reduce granulosa cell apoptosis, and restore ovarian function in autoimmune POI. For example, human bone marrow-derived MSCs have been shown to inhibit pyroptosis and apoptosis of granulosa cells in autoimmune POI models, improving estrous cycles and hormone profiles. These findings highlight the potential of immunomodulatory therapies in managing autoimmune-related POI.³⁰

Iatrogenic injury is a prominent cause of POI, particularly in women undergoing chemotherapy, radiotherapy, or pelvic surgeries. Chemotherapeutic agents such as cyclophosphamide induce DNA damage in ovarian granulosa cells and oocytes, triggering apoptosis and follicular depletion. Radiation therapy similarly causes direct cytotoxic effects on ovarian tissue. Surgical interventions involving the pelvis may disrupt ovarian blood supply or remove ovarian tissue, leading to diminished ovarian reserve. Animal studies have demonstrated that transplantation of MSCs derived from umbilical cord or bone marrow can ameliorate chemotherapy-induced POI by promoting granulosa cell proliferation, reducing apoptosis, and restoring hormone secretion. These stem cells exert their effects through paracrine signaling, anti-inflammatory actions, and activation of survival pathways such as PI3K/AKT. Moreover, MSC-derived extracellular vesicles have been shown to activate integrin-linked kinase (ILK)-PI3K/AKT signaling, further supporting follicular survival and ovarian regeneration after chemotherapeutic injury.^31,32

Environmental and infectious factors also contribute to the pathogenesis of POI by accelerating follicular atresia. Viral infections, notably mumps virus, have been implicated in ovarian inflammation and damage, leading to premature follicle loss. Exposure to environmental toxins such as cigarette smoke and industrial chemicals induces oxidative stress and disrupts ovarian microenvironment homeostasis, promoting follicular apoptosis and fibrosis. Recent research indicates that oxidative stress-induced ferroptosis, a form of regulated cell death driven by iron accumulation and lipid peroxidation, plays a role in granulosa cell loss in POI. Human umbilical cord-derived MSCs have been reported to alleviate chemotherapy-induced POI by suppressing ferritinophagy-mediated ferroptosis in granulosa cells via activation of the NRF2 antioxidant pathway. This mechanistic insight opens new avenues for antioxidant-based adjunct therapies in POI management.^33,34

In summary, the pathogenesis of POI is multifaceted, involving genetic predispositions such as X chromosome abnormalities and gene mutations, autoimmune-mediated follicular destruction, iatrogenic insults from cancer therapies and surgeries, and environmental or infectious insults that exacerbate follicular depletion. Understanding these diverse etiologies is crucial for developing targeted therapeutic strategies. Stem cell therapies, particularly those employing mesenchymal stem cells and their secreted factors, have shown promise in addressing various pathogenic mechanisms by promoting ovarian tissue regeneration, modulating immune responses, and protecting granulosa cells from apoptosis and oxidative damage. Continued research into the molecular pathways underlying POI will facilitate the refinement of these regenerative approaches and improve clinical outcomes for affected women.^35–37

Limitations of Traditional Treatment Methods

Traditional therapeutic approaches for premature ovarian insufficiency (POI) and early menopause primarily focus on symptom management and fertility preservation but face significant limitations in restoring ovarian function and fertility. Hormone replacement therapy (HRT) is widely used to alleviate menopausal symptoms such as hot flashes, osteoporosis, and cardiovascular risks associated with hypoestrogenism. HRT effectively mitigates vasomotor symptoms and reduces bone loss, thereby lowering fracture risk in women with POI or early menopause. However, HRT does not restore ovarian endocrine function or fertility, as it merely supplements deficient hormones without regenerating ovarian follicles or granulosa cells. Hormone therapy is recommended for symptom control and long-term health protection in appropriately selected young women with POI and should be individualized according to contraindications and comorbidities.^5,38–40 HRT does not constitute a regenerative treatment and does not reliably restore endogenous ovarian activity or fertility.

Fertility preservation techniques such as oocyte and embryo cryopreservation represent the mainstay for women desiring future fertility. These methods require ovarian stimulation to retrieve mature oocytes, which are then frozen for later use. While effective, these approaches are only feasible for patients with residual ovarian function and a planned fertility timeline, excluding those with severely diminished ovarian reserve or urgent need for gonadotoxic therapy. Additionally, ovarian stimulation protocols may be contraindicated or impractical in certain clinical scenarios, such as immediate cancer treatment³⁸ Thus, oocyte and embryo cryopreservation do not address the underlying ovarian insufficiency and are limited by patient eligibility and timing constraints.

Ovarian tissue cryopreservation and transplantation have emerged as promising fertility preservation methods, especially for prepubertal girls or women who cannot delay gonadotoxic treatments. This technique involves harvesting and freezing ovarian cortical tissue containing primordial follicles, which can later be autotransplanted to restore endocrine function and fertility. Although successful live births have been reported, the procedure remains experimental with several challenges. Ischemia-reperfusion injury following transplantation leads to substantial follicle loss, reducing graft longevity and efficacy. Moreover, there is a risk of reintroducing malignant cells in patients with cancer, raising safety concerns^38,41 The technical complexity and limited follicular survival currently restrict widespread clinical application. Consequently, ovarian tissue cryopreservation and transplantation have not yet become standard care and require further optimization.

In summary, traditional treatments for POI and early menopause—HRT, oocyte/embryo cryopreservation, and ovarian tissue transplantation—primarily provide symptomatic relief or fertility preservation without restoring intrinsic ovarian function. HRT alleviates menopausal symptoms but does not recover fertility and carries oncologic risks with prolonged use. Cryopreservation techniques are limited by patient selection and do not reverse ovarian insufficiency. Ovarian tissue transplantation is promising but hindered by ischemic damage and safety concerns. These limitations underscore the urgent need for novel regenerative therapies capable of restoring ovarian endocrine function and fertility, such as stem cell-based interventions and bioengineered biomaterials, which are currently under active investigation.³⁸

Theoretical Basis of Stem Cell Therapy for Primary Ovarian Insufficiency

Classification and Characteristics of Stem Cells

Stem cells employed in the treatment of premature ovarian insufficiency (POI) or premature ovarian insufficiency (POI) are diverse, each with distinct origins, differentiation potentials, and clinical applicability. Among these, mesenchymal stem cells (MSCs) represent the most commonly utilized type in POI therapy due to their multipotency, immunomodulatory, and anti-inflammatory properties. MSCs can be isolated from various tissues including bone marrow, adipose tissue, umbilical cord, placenta, and menstrual blood. These cells exhibit the capacity to differentiate into multiple lineages and secrete paracrine factors that promote tissue repair and regeneration. For instance, human umbilical cord mesenchymal stem cells (hUCMSCs) have demonstrated efficacy in restoring ovarian function in animal models of chemotherapy-induced POI by improving estrous cycles, hormone levels, and follicle numbers^42,43 Similarly, placenta-derived MSCs have been shown to restore ovarian function via antioxidant effects, reducing oxidative stress markers and apoptosis in ovariectomized rat models⁴⁴ Menstrual blood-derived MSCs (MenSCs) also exhibit promising therapeutic potential by reducing fibrosis and improving fertility in POI rat models⁴⁵ The therapeutic mechanisms of MSCs involve activation of signaling pathways such as PI3K/AKT, Hippo, and Wnt/β-catenin, which regulate follicular activation, granulosa cell proliferation, and apoptosis inhibition.^46–48

Embryonic stem cells (ESCs), derived from the inner cell mass of blastocysts, possess pluripotent differentiation capacity, enabling them to give rise to all cell types including germ cells. Human ESCs (hESCs) have been considered powerful candidates for POI treatment due to their unlimited self-renewal and differentiation potential. However, their clinical application is limited by ethical concerns and the risk of tumorigenicity. Studies have explored the differentiation of hESCs into oocyte-like cells, but the protocols remain complex and the safety profile uncertain⁴⁹ The ethical debate surrounding the use of ESCs and the potential for teratoma formation have restricted their translational progress in ovarian regenerative medicine.

Induced pluripotent stem cells (iPSCs) offer an alternative pluripotent cell source by reprogramming adult somatic cells to an embryonic-like state. iPSCs circumvent ethical issues associated with ESCs and provide patient-specific cells for autologous therapy. They can differentiate into germ cell lineages and oocyte-like cells in vitro, although the efficiency and safety of reprogramming and differentiation require optimization^49,50 Recent advances include the generation of ovarian support cells (OSCs) from iPSCs to enhance in vitro maturation (IVM) of human oocytes, demonstrating improved maturation rates and developmental competence⁵¹ Despite these advances, challenges remain in ensuring genomic stability, preventing tumorigenicity, and achieving functional oocytes suitable for clinical use.

Ovarian stem cells (OSCs), residing in the ovarian surface epithelium, represent a promising endogenous source for ovarian regeneration. OSCs are characterized by their ability to differentiate directly into oocytes and contribute to neo-oogenesis. These cells express germline markers such as DDX4 and IFITM3 and can be isolated using specific antibodies^52,53 OSCs have been shown to form germ cell nests and oocyte-like structures in vitro, and their transplantation into ovaries can restore folliculogenesis and fertility in animal models⁵⁴ However, the isolation and expansion techniques for OSCs are still immature, and their physiological relevance in adult human ovaries remains under debate due to methodological controversies and difficulties in unequivocal identification^55,56 Moreover, the presence of very small embryonic-like stem cells (VSELs), a pluripotent stem cell population linked developmentally to OSCs, adds complexity to understanding ovarian stem cell biology^57,58 VSELs are small, quiescent cells capable of asymmetric division to generate OSCs, which then proliferate and differentiate into oocytes. Dysfunction of these stem cells has been implicated in ovarian aging, premature ovarian insufficiency, and ovarian pathologies such as polycystic ovary syndrome and cancer.^57,59

In summary, MSCs are currently the most widely studied and applied stem cell type for POI treatment due to their accessibility, safety, and paracrine effects. ESCs and iPSCs offer pluripotent sources with high differentiation potential but face ethical and safety challenges. OSCs and VSELs represent endogenous ovarian stem cell populations with direct oogenic potential, though their clinical application awaits further refinement of isolation and expansion methods and confirmation of their physiological roles. The integration of these stem cell types and understanding their unique characteristics and mechanisms will be crucial for advancing stem cell-based therapies for ovarian insufficiency.

Main Mechanisms of Stem Cell Therapy for Premature Ovarian Insufficiency (POI)

Stem cell therapy has emerged as a promising approach to treat premature ovarian insufficiency (POI) by targeting multiple pathological processes that contribute to ovarian dysfunction. The therapeutic mechanisms of stem cells, particularly mesenchymal stem cells (MSCs), in POI involve complex and interrelated pathways, including paracrine effects, immunomodulation, homing and differentiation, as well as anti-fibrotic and angiogenic actions. These mechanisms collectively contribute to the restoration of ovarian function, folliculogenesis, and hormonal balance, thereby improving fertility outcomes.

Paracrine Effects

One of the primary mechanisms by which stem cells exert therapeutic benefits in POI is through paracrine secretion. MSCs secrete a diverse array of growth factors and cytokines, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), and fibroblast growth factor-2 (FGF-2), which play pivotal roles in ovarian tissue repair. These secreted factors inhibit apoptosis of granulosa cells, which are essential for follicle survival and development, thereby promoting folliculogenesis and ovarian function restoration. For instance, HGF secreted by human umbilical cord mesenchymal stromal cells (hUC-MSCs) activates the PI3K-AKT signaling pathway, a critical pathway for primordial follicle activation, by binding to its receptor c-Met on granulosa cells. This activation enhances the expression of KIT ligand (KITL), further supporting follicle growth and survival⁴⁶ Additionally, the secretome from estrogen receptor-positive placenta-derived MSCs (ER+pcMSCs) contains angiogenin and other cytokines that promote angiogenesis and reduce granulosa cell apoptosis, contributing to ovarian regeneration⁶⁰ These paracrine factors collectively create a microenvironment conducive to follicle survival and development, which is crucial for reversing the ovarian insufficiency characteristic of POI.

Immunomodulation

Immunological dysregulation and local inflammation are significant contributors to ovarian damage in POI, especially in autoimmune-related cases. MSCs possess potent immunomodulatory properties that help restore immune homeostasis within the ovarian microenvironment. They regulate immune cell populations by promoting regulatory T cells (Tregs), suppressing pro-inflammatory T helper 17 (Th17) cells, and modulating macrophage polarization from a pro-inflammatory (M1) to an anti-inflammatory (M2) phenotype. This immunoregulatory effect reduces local ovarian inflammation, thereby protecting ovarian tissue from immune-mediated damage and facilitating tissue repair. For example, in autoimmune-induced POI rat models, transplantation of human umbilical cord MSCs (hUCMSCs) led to normalization of estrous cycles, improved follicular development, and increased serum levels of estradiol and anti-Müllerian hormone (AMH), accompanied by reduced granulosa cell apoptosis and enhanced proliferation, which are indicative of improved immune microenvironment and ovarian function⁶¹ Moreover, the secretome from MSCs contains exosomal miRNAs and cytokines that target genes involved in apoptosis, fibrosis, and immune regulation, further contributing to the immunomodulatory milieu necessary for ovarian recovery.⁶⁰

Homing and Differentiation

The ability of MSCs to home toward injured ovarian tissue has been demonstrated in several preclinical models, but the extent to which durable differentiation into functional ovarian cells contributes to therapeutic benefit remains uncertain. Current evidence more consistently supports paracrine signaling, immunomodulation, anti-apoptotic and antioxidant effects, and angiogenic remodeling. In some experiments, transplanted cells localized predominantly within the ovarian stroma without convincing expression of definitive oocyte or granulosa-cell markers.^62–65 Therefore, claims that MSCs directly replace depleted ovarian cells or generate new functional follicles should be interpreted cautiously.

Anti-Fibrotic and Angiogenic Effects

Ovarian fibrosis and impaired vascularization are hallmark features of POI that contribute to follicular depletion and ovarian dysfunction. MSCs counteract these pathological changes by inhibiting fibrotic signaling pathways, such as transforming growth factor-beta (TGF-β), thereby reducing extracellular matrix deposition and fibrosis within the ovary.^28,66 Concurrently, MSCs promote angiogenesis through the secretion of pro-angiogenic factors like VEGF, which enhance neovascularization and improve ovarian blood supply. Improved vascularization supports follicle survival and function by ensuring adequate nutrient and oxygen delivery. For example, transplantation of human adipose-derived stromal cells (hASCs) in chemotherapy-induced POI mouse models alleviated ovarian stromal alterations and vascular damage, preserved follicle reserves, and prolonged reproductive lifespan⁶⁷ Similarly, placenta-derived MSCs restored ovarian function in ovariectomized rats by upregulating antioxidant factors and reducing oxidative stress, which is closely linked to fibrosis and vascular injury⁴⁴ These anti-fibrotic and angiogenic actions are essential for reconstructing a healthy ovarian microenvironment conducive to follicle regeneration and endocrine function.

Overall, available evidence suggests that MSCs act through interacting paracrine, immunomodulatory, anti-apoptotic, antioxidant, and angiogenic pathways. Direct differentiation may occur in selected experimental settings, but its quantitative contribution to ovarian recovery remains unresolved. These mechanisms should therefore be viewed as experimentally supported hypotheses of varying strength rather than established explanations of clinical benefit.

Stem Cell Therapy in Animal Model Studies

Chemotherapy-Induced POI Models

Collectively, chemotherapy-induced POI models provide proof-of-concept evidence for biological activity, but they should not be regarded as full representations of human POI.⁶⁸ These models usually involve abrupt experimentally induced follicular injury and may not reproduce the genetic, autoimmune, idiopathic, metabolic, or intermittently active phenotypes observed clinically.^69–72 Improvements in follicles, hormone levels, or mating outcomes therefore support biological plausibility but cannot by themselves establish efficacy across the broader POI population.

Natural Aging Models

Natural ovarian aging models, typically involving aged mice or genetically modified strains, serve to investigate the potential of stem cell therapies in delaying ovarian senescence and preserving fertility. These models reflect the gradual decline in follicle quantity and quality due to intrinsic aging processes such as DNA damage accumulation, telomere shortening, and mitochondrial dysfunction. Studies employing aged mice have demonstrated that MSC transplantation can improve ovarian weight, increase follicle reserves, and enhance hormone profiles, thereby mitigating the effects of ovarian aging. For instance, bone marrow-derived MSCs combined with adjunct therapies like royal jelly have been shown to restore ovarian tissue function in polycystic ovary syndrome (PCOS) rat models by regulating inflammation, angiogenesis, and oocyte quality markers such as BMP15 and GDF9, which are also relevant in aging-related ovarian decline⁷³ Furthermore, MSCs delay ovarian aging through antioxidative effects, promotion of cell proliferation, inhibition of apoptosis, and modulation of inflammatory responses, as evidenced by improved ovarian morphology and follicular development in aged animal models⁷⁴ The paracrine secretion of cytokines and growth factors by MSCs plays a pivotal role in these processes, enhancing the ovarian microenvironment and supporting follicular survival. Although fewer studies have focused explicitly on natural aging models compared to chemotherapy-induced models, the available data underscore the promise of MSC-based interventions in extending ovarian lifespan and function, potentially translating into therapeutic strategies for age-related infertility.

Gene Knockout Models

Genetic knockout models, such as mice harboring mutations in key ovarian regulatory genes like bone morphogenetic protein 15 (BMP15) or growth differentiation factor 9 (GDF9), provide valuable insights into the pathophysiology of hereditary POI and the reparative effects of stem cell therapies. These models mimic genetic causes of POI characterized by impaired folliculogenesis and premature depletion of the ovarian reserve. Although research in this area remains limited, preliminary studies suggest that MSC transplantation may ameliorate ovarian dysfunction in gene knockout models by restoring expression of critical oocyte-secreted factors and improving follicular development. For example, BMP15 and GDF9 protein levels, which are significantly reduced in PCOS and potentially in genetic POI models, have been shown to increase following MSC treatment, indicating enhanced oocyte quality and follicular maturation⁷³ The mechanisms likely involve MSC-mediated modulation of the ovarian niche, including anti-inflammatory effects, angiogenesis promotion, and inhibition of apoptosis. However, the scarcity of studies specifically addressing MSC therapy in genetic POI models highlights a gap in current research. Future investigations are warranted to elucidate the efficacy and underlying molecular pathways of stem cell interventions in hereditary POI, which could pave the way for personalized regenerative therapies targeting genetic etiologies of ovarian failure.

Preclinical Study of Mesenchymal Stem Cell Therapy for Primary Ovarian Insufficiency

Bone Marrow Mesenchymal Stem Cells (BMSCs) Applications

Bone marrow mesenchymal stem cells (BMSCs) have emerged as a pivotal therapeutic tool in the treatment of premature ovarian insufficiency (POI), particularly in animal models induced by chemotherapeutic agents such as cyclophosphamide. In cyclophosphamide-induced POI rat models, intravenous administration of BMSCs has been shown to significantly upregulate the expression of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) within ovarian tissues. These growth factors are critical for angiogenesis and tissue repair, facilitating the restoration of ovarian microenvironment and follicular development. The enhanced expression of VEGF and HGF correlates with a marked reduction in granulosa cell apoptosis, which is a key pathological feature of POI. Consequently, serum estradiol (E2) levels increase, reflecting improved ovarian endocrine function, while follicle-stimulating hormone (FSH) levels decrease, indicating a reversal of the hypergonadotropic state characteristic of ovarian insufficiency^35,48 This therapeutic effect is mediated not only by the differentiation potential of BMSCs into granulosa-like cells but also through their paracrine secretion of cytokines and growth factors that modulate the ovarian niche.

Beyond direct cell replacement, BMSCs exert significant effects via their secreted extracellular vesicles, particularly exosomes, which carry microRNAs (miRNAs) such as miR-21 and miR-146a. These miRNAs play crucial roles in suppressing apoptosis and inflammatory responses within ovarian cells. For instance, miR-21 has been implicated in the inhibition of granulosa cell apoptosis by targeting pro-apoptotic genes, while miR-146a modulates inflammatory signaling pathways, thereby creating a more favorable environment for follicular survival and development. The delivery of these miRNAs through BMSC-derived exosomes activates intracellular signaling cascades such as the PI3K/AKT pathway, which promotes cell survival and proliferation. This mechanism has been validated in various preclinical studies demonstrating that BMSC exosomes can restore estrous cyclicity, increase antral follicle counts, and normalize hormone levels in POI models^75–77 The paracrine action of BMSCs via exosomal miRNAs thus represents a cell-free therapeutic strategy with potential advantages in safety and ease of administration.

Regarding the delivery methods of BMSCs, local injection into the ovarian hilum region has been explored to enhance the homing efficiency of transplanted cells. This targeted approach increases the concentration of BMSCs in the ovarian microenvironment, potentially amplifying their regenerative effects. However, local injection carries inherent risks such as puncture-induced tissue damage and inflammation, which may compromise ovarian integrity. Therefore, optimizing the injection route is critical to maximize therapeutic efficacy while minimizing adverse effects. Intravenous administration remains a less invasive alternative, with evidence showing that BMSCs can home to injured ovarian tissue, albeit with lower localization efficiency compared to direct ovarian injection^65,78 Strategies to improve homing, such as preconditioning of BMSCs or use of biomaterial scaffolds, are under investigation to overcome these limitations.

In summary, BMSCs contribute to ovarian repair in POI models through multifaceted mechanisms including secretion of angiogenic factors (VEGF, HGF), delivery of anti-apoptotic and anti-inflammatory miRNAs via exosomes, and potential differentiation into ovarian cell types. The modulation of granulosa cell apoptosis and enhancement of follicular development lead to restoration of hormonal balance and fertility. While local ovarian injection of BMSCs improves cell homing, it requires careful optimization to avoid procedural injury. Continued research into the molecular pathways involved and delivery techniques will be essential to translate BMSC therapy into clinical practice for POI patients.^{35,48,65,75,76,78}

Application of Umbilical Cord Mesenchymal Stem Cells (UC-MSCs)

Umbilical cord mesenchymal stem cells (UC-MSCs) have emerged as a highly promising therapeutic option for premature ovarian insufficiency (POI) due to their abundant availability, non-invasive collection methods, and low immunogenicity. These characteristics make UC-MSCs particularly attractive for clinical applications in ovarian regenerative medicine. In various POI animal models, especially in mice induced by chemotherapy agents such as cyclophosphamide and busulfan, transplantation of UC-MSCs has demonstrated significant restoration of ovarian morphology and function. Notably, treated ovaries exhibit increased volume and a marked rise in the number of primordial and primary follicles, indicating enhanced folliculogenesis and ovarian reserve recovery. This regenerative effect is accompanied by improved hormonal profiles, including elevated estradiol (E2) and anti-Müllerian hormone (AMH) levels, and reduced follicle-stimulating hormone (FSH), reflecting the restoration of endocrine function and follicular activity.^79–81

Mechanistically, UC-MSCs facilitate ovarian repair through activation of key intracellular signaling pathways that govern cell survival, proliferation, and differentiation. Among these, the PI3K/Akt pathway plays a central role in promoting granulosa cell proliferation and follicle development, while the Wnt/β-catenin signaling cascade contributes to follicular growth and maturation. Activation of these pathways by UC-MSCs leads to enhanced granulosa cell viability and follicular progression, which are critical for restoring ovarian function. Concurrently, UC-MSCs exert antioxidative effects by suppressing oxidative stress-induced damage within the ovarian microenvironment. This is evidenced by decreased reactive oxygen species (ROS) levels and downregulation of proinflammatory cytokines such as interleukin-6 (IL-6), IL-18, and tumor necrosis factor-alpha (TNF-α), which are known to exacerbate ovarian injury and follicular atresia. The inhibition of oxidative stress and inflammation contributes to the preservation of follicular integrity and prevention of granulosa cell apoptosis, thereby supporting ovarian regeneration.^81–83

A distinctive advantage of UC-MSCs over other mesenchymal stem cell sources, such as bone marrow-derived MSCs (BMSCs), lies in their potent paracrine activity. UC-MSCs secrete a rich repertoire of bioactive factors, including cytokines, growth factors, and extracellular vesicles (EVs), which mediate tissue repair and immunomodulation. Recent studies have highlighted that the paracrine effects of UC-MSCs are more robust than those of BMSCs, particularly through the secretion of EVs that encapsulate proteins, lipids, and microRNAs critical for intercellular communication. These EVs have been shown to play a pivotal role in ovarian function restoration by delivering regenerative signals to damaged ovarian cells, modulating the local immune response, and promoting angiogenesis. For instance, UC-MSC-derived exosomes can reduce inflammation, inhibit matrix metalloproteinases, and enhance extracellular matrix remodeling, thereby creating a conducive niche for follicular recovery. The use of EVs as a cell-free therapeutic modality also offers advantages in terms of safety, storage, and standardization, potentially overcoming some limitations associated with direct stem cell transplantation.^82,84,85

Furthermore, the therapeutic efficacy of UC-MSCs can be augmented by optimizing their culture conditions and delivery methods. Three-dimensional (3D) spheroid cultures of UC-MSCs have demonstrated superior resistance to oxidative stress and enhanced secretion of protective factors compared to traditional monolayer cultures. This 3D configuration improves cell survival post-transplantation and amplifies the anti-apoptotic and anti-autophagic effects on ovarian granulosa cells, which are crucial for follicle maintenance. Additionally, repeated UC-MSC transplantation has been shown to yield better outcomes in ovarian function recovery than single-dose administration, as evidenced by increased follicle counts, hormone levels, and fertility rates in POI models. These findings underscore the importance of dosing strategies and cell preparation techniques in maximizing the regenerative potential of UC-MSCs.^79,83

Preclinical studies of UC-MSCs have generally not identified major acute toxicities under the tested experimental conditions, but this should not be interpreted as proof of long-term clinical safety. Safety may depend on cell source, manufacturing, dose, route, repeated administration, and recipient characteristics. Long-term surveillance remains necessary to assess tumorigenicity, ectopic tissue formation, immune effects, vascular complications, and delayed adverse events.^86–89

In summary, UC-MSCs represent a promising and versatile cell source for the treatment of premature ovarian insufficiency. Their ability to enhance follicular development, restore hormonal balance, and mitigate oxidative and inflammatory damage through activation of PI3K/Akt and Wnt/β-catenin pathways, combined with potent paracrine effects mediated by extracellular vesicles, underpins their therapeutic efficacy. Ongoing research focusing on optimizing cell culture, delivery methods, and understanding molecular mechanisms will further advance the clinical translation of UC-MSC-based therapies for ovarian regeneration.

Application of Adipose-Derived Mesenchymal Stem Cells (ADSCs)

Adipose-derived mesenchymal stem cells (ADSCs) have garnered significant attention in regenerative medicine due to their relative ease of harvest, abundance, and potent therapeutic properties. ADSCs can be obtained in large quantities from adipose tissue with minimally invasive procedures, making them highly accessible compared to other stem cell sources such as bone marrow. In the context of premature ovarian insufficiency (POI) or premature ovarian insufficiency (POI), animal studies have demonstrated that transplantation of ADSCs into POI rat models leads to notable improvements in ovarian histology and function. Specifically, ADSC transplantation has been shown to alleviate ovarian stromal fibrosis, increase vascular density, and reduce follicular atresia, thereby contributing to the restoration of ovarian reserve and function. These effects are critical because fibrosis and follicle loss are hallmark pathological features of POI that compromise ovarian endocrine and reproductive capabilities.⁹⁰

The therapeutic mechanisms of ADSCs in ovarian repair extend beyond direct cell replacement. ADSCs exert paracrine effects by secreting anti-inflammatory cytokines such as interleukin-10 (IL-10) and transforming growth factor-beta 1 (TGF-β1), which modulate the ovarian microenvironment. One key immunomodulatory action is the regulation of macrophage polarization: ADSCs promote the shift of macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. This polarization shift reduces ovarian inflammation, which is a significant contributor to follicular damage and ovarian dysfunction in POI. By dampening the inflammatory milieu, ADSCs create a more conducive environment for follicular survival and growth, thus enhancing ovarian recovery.⁹¹

Comparative studies between ADSCs and bone marrow-derived mesenchymal stem cells (BMSCs) reveal that ADSCs may possess superior angiogenic potential. Angiogenesis is vital for ovarian tissue repair as it restores blood supply, delivering oxygen and nutrients essential for follicle development and hormone synthesis. ADSCs secrete higher levels of pro-angiogenic factors such as vascular endothelial growth factor (VEGF), which stimulate neovascularization more effectively than BMSCs. This enhanced angiogenic capacity positions ADSCs as a promising candidate for therapies aimed at reversing ovarian insufficiency. However, despite these advantages, the long-term safety profile of ADSC transplantation remains incompletely characterized. Concerns include potential risks of ectopic tissue formation, immunogenicity, and tumorigenicity, which necessitate rigorous preclinical and clinical evaluations before widespread clinical application.⁹⁰

Beyond their cellular effects, ADSCs also release extracellular vesicles (EVs) and exosomes that carry bioactive molecules capable of modulating recipient cell behavior. These vesicles can replicate many of the therapeutic benefits of ADSCs, including immunomodulation, anti-fibrotic effects, and promotion of angiogenesis, while potentially reducing risks associated with cell transplantation. For instance, ADSC-derived exosomes have been shown to attenuate inflammation and oxidative stress, enhance vascularization, and promote tissue regeneration in various models. This cell-free therapeutic approach is gaining momentum as a safer and more controllable alternative to direct stem cell transplantation.⁹²

In summary, ADSCs offer a multifaceted therapeutic approach for POI by mitigating fibrosis, enhancing angiogenesis, and modulating the inflammatory microenvironment through cytokine secretion and macrophage polarization. Their ease of harvest and potent paracrine effects make them attractive candidates for regenerative therapies targeting ovarian insufficiency. Nonetheless, further studies are essential to establish their long-term safety and optimize delivery methods to maximize therapeutic efficacy while minimizing potential adverse effects.

Clinical Progress of Stem Cell Therapy for Primary Ovarian Insufficiency

Clinical Trial Design and Results (Table 2)

Table 2.

Preclinical Strategies of Stem Cell Therapy for Premature Ovarian Insufficiency: Representative Models, Interventions, Reported Outcomes, and Key Limitations

Preclinical Strategy	Representative Model	Intervention	Main Reported Outcomes	Key Limitation
BMSCs	Chemotherapy-induced POI rodents	Systemic/local BMSC administration	Hormonal, follicular, angiogenic and anti-apoptotic changes	Dose/route heterogeneity; direct differentiation uncertain
UC-MSCs	Cyclophosphamide/busulfan models	Single/repeated UC-MSC transplantation	Ovarian morphology, hormones, follicles, reproductive parameters	Acute toxicant models may not represent heterogeneous human POI
ADSCs	Chemotherapy/fibrosis-dominant models	Local ADSC administration	Reduced fibrosis; improved vascularization and follicular parameters	Long-term safety and optimal cell preparation uncertain
MSC-derived EVs	Chemotherapy-induced models	Exosome/EV administration	Anti-inflammatory, antioxidant and anti-apoptotic effects	Predominantly preclinical; potency/composition require standardization

Clinical evidence remains substantially less mature than the preclinical literature. Registered trials include studies of UC-MSCs, BMSCs, adipose-derived cells, and scaffold-assisted transplantation; however, trial registration indicates planned investigation rather than demonstrated efficacy. For example, NCT03033277 is a non-randomized clinical study of UC-MSC transplantation, whereas NCT02644447 evaluated allogeneic UC-MSCs with an injectable collagen scaffold. Registry status and peer-reviewed outcome reports should therefore be distinguished when summarizing clinical evidence.

A published clinical analysis of UC-MSC transplantation reported menstrual recovery and changes in ovarian biomarkers in a subset of treated women; however, the study was non-randomized and participants also received hormone replacement therapy, limiting causal attribution to the cell intervention.⁹³ These findings support feasibility and a possible biological signal but do not establish restoration of ovarian reserve or fertility. Similarly, small studies of intraovarian BMSCs, ADSCs, and menstrual blood-derived MSCs have reported changes in endocrine or menstrual outcomes, but their uncontrolled or non-randomized designs and small samples substantially limit inference about comparative efficacy.^94–96

Importantly, the clinical endpoint hierarchy should be explicit. AMH, FSH, estradiol, AFC, ovarian volume, and menstrual recovery are surrogate or intermediate outcomes. They may indicate altered ovarian activity, but they do not demonstrate de novo follicle formation, durable restoration of the primordial follicle pool, ovulation, or fertility. Pregnancy reports also require careful interpretation because intermittent ovarian activity can occur in POI and conception may involve IVF, embryo transfer, donor oocytes, or other concurrent interventions. Live birth attributable to the stem-cell intervention remains the most clinically meaningful reproductive endpoint but is currently supported by very limited evidence.

Clinical studies also differ substantially in cell source, dose, route, concomitant treatment, eligibility criteria, comparator groups, and follow-up. These differences preclude direct comparison of reported effects and make it inappropriate to describe the current evidence as establishing clinical effectiveness. Overall, human studies should be considered preliminary and heterogeneous, with their principal contribution at present being assessment of feasibility, safety signals, and potential biological activity rather than confirmation of therapeutic efficacy.

Some registered studies evaluate fertility or pregnancy outcomes as primary endpoints, whereas others focus mainly on hormonal or imaging biomarkers.⁹⁷ Such protocols are important for the evolution of the field, but registration alone cannot be interpreted as evidence of efficacy. Future controlled trials should prespecify clinically meaningful reproductive endpoints, standardized ovarian biomarkers, appropriate comparator groups, and sufficiently long follow-up.

Mechanistically, MSCs exert their therapeutic effects through multiple pathways, including paracrine secretion of growth factors, immunomodulation, and promotion of angiogenesis within the ovarian niche. For example, bone marrow-derived MSCs have been shown to regulate the TGF-β1/Smad2/3 signaling pathway and enhance VEGF expression, thereby reducing ovarian stromal fibrosis and improving the angiogenic microenvironment critical for follicular development⁹⁸ Additionally, MSC-derived exosomes have demonstrated efficacy in mitigating granulosa cell pyroptosis and oxidative stress, further supporting ovarian function recovery.⁷⁶

Despite these advances, challenges remain in standardizing stem cell preparations, optimizing dosing regimens, and ensuring long-term safety. The heterogeneity of MSC populations, influenced by tissue source and donor variability, necessitates rigorous characterization and quality control to enhance therapeutic consistency⁹⁹ Moreover, the limited sample sizes and short follow-up durations in current trials highlight the need for larger, multicenter randomized controlled studies with standardized outcome measures to validate efficacy and safety comprehensively.¹⁰⁰

In summary, clinical trials to date provide encouraging evidence that MSC-based therapies, particularly those utilizing UC-MSCs and BMSCs, can improve ovarian endocrine function and partially restore menstruation in POI patients with a favorable safety profile. However, translating these biological improvements into meaningful fertility outcomes remains a critical goal. Future research should focus on refining cell sourcing, delivery techniques, and combination therapies, alongside long-term monitoring, to fully realize the potential of stem cell therapy in treating premature ovarian insufficiency.

Optimization of Stem Cell Sources and Administration Routes (Table 3)

Table 3.

Representative Early Human Studies of Stem Cell Therapy for Premature Ovarian Insufficiency: Study Design, Cell Source and Administration Route, Main Endpoints, and Critical Interpretation

Study/Evidence	Design / N	Cell Source / Route	Main Endpoint	Critical Interpretation
Yan et al, 202093	Non-randomized; 61 initially treated	UC-MSCs; intraovarian injection	Hormones, follicular parameters, menstruation	Concomitant HRT and lack of randomized comparator limit causal inference
Igboeli et al94	Small clinical report	Autologous BMSCs; intraovarian	E2, follicles, pregnancy	Highly limited by small sample and design
ADSC study95	Phase I, non-randomized	ADSCs; intraovarian	Menstruation, FSH, safety	Feasibility signal; uncontrolled
Men-MSC study96	Early clinical study	Menstrual blood-derived MSCs; intraovarian	Menstruation, AMH	Menstrual recovery reported; AMH change not significant
NCT02644447	Registered Phase I/II; 23 planned	Allogeneic UC-MSCs ± collagen scaffold	Safety and ovarian-function endpoints	Registration/registry status does not demonstrate efficacy
NCT03033277	Registered Phase I/II; 320 planned	UC-MSCs; intraovarian	Ovarian-function and fertility-related outcomes	Registry evidence; no efficacy inference from registration alone

The optimization of stem cell sources and administration routes is pivotal in enhancing the therapeutic efficacy and safety of stem cell therapy for premature ovarian insufficiency (POI). Autologous stem cells, such as autologous bone marrow-derived mesenchymal stem cells (BMSCs), offer the advantage of minimizing immune rejection due to their origin from the patient’s own tissues. Clinical reports have demonstrated that intraovarian injection of autologous BMSCs can lead to significant improvements in ovarian volume, estrogen production, and menstrual recovery in POI patients, with sustained effects observed up to one year post-treatment and no reported adverse events, underscoring their safety and potential for clinical application⁹⁴ However, a critical limitation of autologous stem cells is the potential impairment of their function in patients with POI, as the disease process or prior treatments may compromise the quality and regenerative capacity of the patient’s own stem cells¹⁰¹ This necessitates consideration of alternative sources.

Allogeneic stem cells, particularly umbilical cord-derived mesenchymal stem cells (UC-MSCs), have emerged as promising candidates due to their abundant availability, low immunogenicity, and potent regenerative properties. UC-MSCs have been shown to improve ovarian function and fertility outcomes in both preclinical and clinical settings. For instance, UC-MSC transplantation via transvaginal ultrasound-guided intraovarian injection in POI patients resulted in increased antral follicle count, higher numbers of oocytes retrieved during IVF cycles, and improved embryo freezing rates compared to controls, indicating enhanced ovarian reserve and function¹⁰² Nevertheless, the use of allogeneic cells requires careful consideration of human leukocyte antigen (HLA) matching and potential immunosuppressive strategies to mitigate immune rejection. Preclinical toxicity studies in rats have revealed that high doses of intraovarian UC-MSC injections can induce inflammatory responses in ovarian and oviduct tissues and increase mortality, highlighting the importance of dose optimization and safety evaluation.¹⁰³

The route of stem cell administration significantly influences cell homing efficiency, engraftment, and therapeutic outcomes. Common administration routes include intravenous injection, ovarian artery injection, laparoscopic-guided intraovarian injection, and ultrasound-guided transvaginal intraovarian injection. Intravenous injection is minimally invasive and allows systemic delivery; however, it may result in lower homing efficiency to the ovaries due to cell entrapment in other organs such as the lungs and liver⁴⁴ In contrast, local administration directly into the ovary, either via laparoscopic or ultrasound guidance, enhances the concentration of stem cells at the target site, improving engraftment and therapeutic efficacy. For example, intraovarian injection of human bone marrow-derived MSCs in chemotherapy-induced POI mouse models restored hormone production and folliculogenesis more effectively than systemic administration¹⁰⁴ However, local injection techniques are more complex and invasive, requiring specialized equipment and expertise, which may limit their widespread clinical adoption.

Emerging delivery strategies aim to further optimize stem cell retention and function. Scaffold-based local delivery systems, such as hydrogel encapsulation, have been developed to prolong stem cell survival and enhance paracrine effects. Studies using bioinspired scaffolds for local delivery of embryonic stem cell-derived mesenchymal progenitor cells demonstrated improved ovarian function recovery in chemotherapy-induced POI models compared to systemic administration, suggesting that scaffold-mediated local delivery may represent a superior approach¹⁰⁵ Additionally, three-dimensional culture of UC-MSCs to produce extracellular vesicles combined with extracellular matrix gels has shown enhanced therapeutic effects on ovarian function restoration in rat models, indicating that cell-free therapies with optimized delivery vehicles may also be viable alternatives.¹⁰⁶

Regarding dosing regimens, single stem cell injections have shown limited duration of therapeutic effects in some studies, with engrafted cells gradually diminishing over weeks post-transplantation¹⁰⁴ Repeated administration protocols, such as monthly injections over three months, have been proposed to sustain ovarian function improvements. For instance, multiple intravenous injections of human amniotic mesenchymal stem cells (hAMSCs) at defined doses were well tolerated in animal models and effectively restored ovarian function, suggesting that repeated dosing can enhance efficacy while maintaining safety¹⁰⁷ Nonetheless, the long-term safety of repeated stem cell administrations requires thorough evaluation to preclude potential adverse effects such as tumorigenicity or immune sensitization.

In summary, the choice between autologous and allogeneic stem cells involves balancing immunological compatibility against cell quality and availability. Local administration routes, particularly intraovarian injections guided by laparoscopy or ultrasound, improve stem cell homing and therapeutic outcomes but are technically demanding. Scaffold-based delivery systems and repeated dosing regimens offer promising avenues to enhance stem cell retention and efficacy. Future research should focus on standardizing stem cell sources, optimizing administration routes and dosing schedules, and rigorously assessing long-term safety to facilitate the translation of stem cell therapies into effective clinical treatments for POI. Network meta-analyses suggest that UC-MSCs combined with intraovarian or tail vein injection may provide superior therapeutic benefits compared to other sources and routes, reinforcing the need for well-designed clinical trials to validate these findings.²³

Clinical Efficacy Evaluation Indicators

Biomarkers of Ovarian Endocrine Activity: FSH, LH, E2, AMH, and Inhibin B

Hormonal parameters are useful for monitoring ovarian endocrine activity, but they should be interpreted as intermediate outcomes rather than direct measures of fertility. Changes in FSH, E2, AMH, or inhibin B may indicate altered follicular activity or endocrine feedback, but they cannot independently demonstrate restoration of the primordial follicle pool or reproductive competence.^22,23 Clinical studies should therefore report these measures together with menstrual status, ovulation, follicular development, and reproductive outcomes.

Follicular and Morphological Outcomes: AFC and Ovarian Volume

AFC and ovarian volume obtained by transvaginal ultrasound provide useful information on ovarian morphology and follicular activity.¹⁰⁸ However, an increase in AFC or ovarian volume should not automatically be described as ovarian regeneration. These parameters can fluctuate and are influenced by measurement technique, hormonal exposure, and residual ovarian activity.¹⁰⁹ In preclinical studies, histological follicle counts provide complementary information, but even these findings cannot establish true de novo folliculogenesis without rigorous lineage-tracing or equivalent evidence.¹¹⁰

Reproductive Outcomes: Ovulation, Pregnancy, Ongoing Pregnancy, and Live Birth

Reproductive outcomes are the most clinically relevant endpoints. Natural ovulation, spontaneous conception, ongoing pregnancy, and especially live birth provide stronger evidence of functional reproductive benefit than surrogate ovarian biomarkers. Pregnancy after stem-cell treatment should nevertheless be interpreted according to whether conception was spontaneous or occurred after IVF, embryo transfer, donor oocytes, or other fertility treatment. At present, the number of well-documented reproductive outcomes directly attributable to stem-cell treatment remains small. Reproductive efficacy should therefore be regarded as incompletely established rather than as a demonstrated consequence of ovarian biomarker improvement.

In-Depth Analysis of the Mechanisms of Stem Cell Therapy for Primary Ovarian Insufficiency

Paracrine Factors and Signaling Pathways

Mesenchymal stem cells (MSCs) exert therapeutic effects on premature ovarian insufficiency (POI) predominantly through paracrine mechanisms involving the secretion of growth factors, cytokines, and extracellular vesicles (EVs), which activate key intracellular signaling pathways to promote ovarian follicle survival and granulosa cell function.¹¹¹ Among these factors, vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) secreted by MSCs have been shown to activate the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway, which plays a critical role in inhibiting apoptosis of granulosa cells and enhancing follicular survival. For instance, human umbilical cord MSC-derived extracellular vesicles (UC-MSC-EVs) carrying clusterin (CLU) protein activate the PI3K/Akt pathway, reducing granulosa cell apoptosis and promoting proliferation, thereby restoring ovarian function in chemotherapy-induced POI models¹¹² Similarly, insulin-like growth factor 1 (IGF-1), another paracrine factor secreted by MSCs, stimulates granulosa cell proliferation via the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, further supporting follicular development and ovarian reserve maintenance.¹¹³

Extracellular vesicles, particularly exosomes derived from MSCs, carry a repertoire of microRNAs (miRNAs) that modulate gene expression in recipient ovarian cells, influencing apoptosis, inflammation, and cellular proliferation. Notably, miRNAs such as miR-21, miR-146a, and miR-17-5p have been identified within MSC-derived exosomes and are implicated in targeting genes like phosphatase and tensin homolog (PTEN) and tumor necrosis factor receptor-associated factor 6 (TRAF6), which are key regulators of apoptosis and inflammatory responses. For example, miR-21-enriched exosomes from bone marrow MSCs inhibit granulosa cell pyroptosis by targeting Mucosa-Associated Lymphoid Tissue Lymphoma Translocation Protein 1 (MALT1) and modulating the NF-κB signaling pathway, thereby repairing chemotherapy-induced ovarian damage¹¹⁴ Additionally, miR-17-5p delivered by human umbilical cord MSC-derived exosomes suppresses SIRT7 expression, leading to reduced oxidative stress and enhanced granulosa cell proliferation in POI models¹¹⁵ These miRNAs collectively contribute to the anti-apoptotic and anti-inflammatory milieu necessary for ovarian tissue regeneration.

Another critical paracrine factor secreted by MSCs is stromal cell-derived factor 1 alpha (SDF-1α), which binds to the C-X-C chemokine receptor type 4 (CXCR4) on endogenous stem cells, facilitating their homing to injured ovarian tissue.¹¹⁶ This chemotactic axis establishes a positive feedback loop that enhances the recruitment and retention of endogenous stem cells, amplifying tissue repair and follicular regeneration. Studies have demonstrated that MSC-secreted SDF-1α promotes endogenous stem cell migration, contributing to improved ovarian microenvironment and function post-chemotherapy³⁵ This homing mechanism is essential for sustaining the reparative process initiated by exogenous MSC therapy.

Furthermore, MSC-derived exosomes modulate additional signaling pathways such as Hippo, SMAD, and SIRT1/FOXO1, which are involved in cellular proliferation, differentiation, and oxidative stress responses. For instance, human placental MSC-derived exosomes upregulate IGF-1 and VEGF expression, reduce granulosa cell apoptosis, and regulate the follicular microenvironment through these pathways, enhancing ovarian function in POI models¹¹³ Hypoxia-preconditioned MSC exosomes enriched with circular RNAs (circRNAs) like circPTP4A2 stabilize Y-box binding protein 1 (YBX1), augmenting antioxidative capacity and mitochondrial function in granulosa cells via the PI3K/Akt axis, further supporting follicular survival.¹¹⁶

Collectively, the paracrine secretion of growth factors such as VEGF, HGF, and IGF-1, the delivery of miRNAs via exosomes targeting apoptotic and inflammatory genes, and the chemotactic recruitment of endogenous stem cells through the SDF-1α/CXCR4 axis constitute a multifaceted network of signaling pathways. These pathways synergistically inhibit granulosa cell apoptosis, promote proliferation, and restore ovarian microenvironment homeostasis, thereby facilitating follicular survival and ovarian function recovery in premature ovarian insufficiency. The elucidation of these mechanisms not only advances our understanding of MSC-based therapies but also provides a foundation for developing targeted, cell-free therapeutic strategies for POI.^35,117–125

Immunomodulation and Microenvironment Remodeling

Mesenchymal stem cells (MSCs) have emerged as pivotal agents in the treatment of premature ovarian insufficiency (POI) and premature ovarian insufficiency (POI) due to their multifaceted immunomodulatory properties and ability to remodel the ovarian microenvironment. A key mechanism by which MSCs exert therapeutic effects is through the secretion of anti-inflammatory cytokines such as interleukin-10 (IL-10), transforming growth factor-beta 1 (TGF-β1), and prostaglandin E2 (PGE2). These factors collectively suppress T cell proliferation and inhibit the differentiation of pro-inflammatory T helper 17 (Th17) cells, while promoting the expansion of regulatory T cells (Tregs), which are crucial for maintaining immune tolerance and preventing autoimmune-mediated ovarian damage. For instance, studies have demonstrated that umbilical cord-derived MSCs (UCMSCs) expressing heme oxygenase-1 (HO-1) can restore ovarian function in POI mice by upregulating IL-10 and increasing circulating CD8+CD28- T cells, highlighting the immunosuppressive milieu fostered by MSCs that mitigates ovarian autoimmunity¹²⁶ Furthermore, MSCs engineered to express pigment epithelium-derived factor (PEDF) have been shown to enhance Treg populations via the Tim-3/Gal-9 immune checkpoint pathway, thereby reducing interferon-gamma (IFN-γ) levels and promoting follicular survival in primary ovarian failure models.¹²⁷

In addition to modulating T cell subsets, MSCs influence macrophage polarization within the ovarian microenvironment. They promote a shift from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, which is associated with tissue repair and fibrosis resolution. Placental MSCs (PMSCs), for example, have been reported to ameliorate NLRP3 inflammasome-induced ovarian insufficiency by inducing M2 macrophage polarization, leading to decreased levels of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), and increased anti-inflammatory cytokines including IL-4 and IL-10¹²⁸ This macrophage reprogramming reduces ovarian inflammation and fibrosis, thereby improving follicular function and ovarian reserve. Similarly, chito-oligosaccharides have been found to synergize with macrophages to enhance ovarian germ stem cell proliferation by modulating inflammatory cytokines, further underscoring the critical role of macrophage-mediated immune regulation in ovarian microenvironment remodeling.¹²⁹

Natural killer (NK) cells and dendritic cells (DCs) also contribute to ovarian immune homeostasis and follicular integrity. MSCs have been shown to suppress NK cell cytotoxic activity and inhibit DC maturation, thereby attenuating immune-mediated follicular damage. Although direct evidence in ovarian contexts is limited, the broad immunosuppressive effects of MSCs on innate immune cells are well documented in other tissue systems and likely translate to the ovarian niche. For example, human amniotic epithelial cells (hAECs), which share immunomodulatory properties with MSCs, have demonstrated efficacy in preventing infertility and miscarriage by regulating maternal immune tolerance, suggesting similar mechanisms may operate in ovarian immune regulation¹³⁰ Moreover, exosomes derived from MSCs carry immunoregulatory molecules that can modulate local immune responses, reduce inflammation, and promote tissue repair, as evidenced by improved ovarian function and reduced granulosa cell apoptosis in chemotherapy-induced POI models following MSC-derived exosome treatment.

The remodeling of the ovarian microenvironment by MSCs extends beyond immune modulation to include attenuation of oxidative stress, fibrosis, and restoration of angiogenesis, all of which are interlinked with immune homeostasis. MSCs secrete a variety of paracrine factors that reduce oxidative damage and inhibit fibrotic pathways such as the TGF-β1/Smad3 signaling axis, thereby reversing ovarian tissue fibrosis and improving follicular development¹³¹ Additionally, MSCs enhance angiogenesis through upregulation of vascular endothelial growth factor (VEGF) and insulin-like growth factor 1 (IGF-1), which supports follicular survival and growth¹³² These effects collectively contribute to the restoration of a supportive ovarian niche conducive to folliculogenesis and endocrine function.

In summary, MSCs orchestrate a complex immunoregulatory network within the ovarian microenvironment by secreting anti-inflammatory cytokines that suppress effector T cell responses and promote Treg expansion, modulating macrophage polarization towards an M2 phenotype, and inhibiting the activation of NK cells and dendritic cells. These immunomodulatory actions, coupled with the secretion of trophic factors that mitigate oxidative stress, fibrosis, and vascular insufficiency, culminate in the remodeling of the ovarian microenvironment. This remodeling alleviates autoimmune and inflammatory insults, restores follicular viability, and ultimately improves ovarian function and fertility outcomes in POI/POI models. Ongoing research continues to elucidate the precise molecular pathways involved and optimize MSC-based therapies to harness these immunomodulatory and microenvironmental remodeling capabilities for clinical application.

Anti-Apoptotic and Anti-Oxidative Stress Effects

Mesenchymal stem cells (MSCs) have demonstrated significant potential in mitigating premature ovarian insufficiency (POI) and ovarian aging by exerting anti-apoptotic and anti-oxidative stress effects, which are critical in preserving ovarian function. One key mechanism involves the regulation of the mitochondrial apoptosis pathway through modulation of the Bcl-2/Bax ratio. MSCs upregulate the anti-apoptotic protein Bcl-2 while downregulating the pro-apoptotic protein Bax, thereby inhibiting mitochondrial outer membrane permeabilization and subsequent activation of caspases that lead to granulosa cell and oocyte apoptosis. This modulation reduces follicular atresia and supports follicular survival, as evidenced by decreased TUNEL-positive cells and cleaved PARP expression in ovarian tissues following MSC transplantation in animal models of chemotherapy-induced POI^64,133,134 The anti-apoptotic effect is further enhanced by MSC-derived exosomes and apoptotic vesicles, which deliver bioactive molecules such as miRNAs (eg, miR-21-5p) that suppress pro-apoptotic genes like PTEN, thereby promoting cell survival and follicular development.^76,134

In addition to anti-apoptotic actions, MSCs contribute to the reduction of oxidative stress in the ovarian microenvironment, a major contributor to ovarian aging and follicular depletion. MSCs secrete antioxidant enzymes including superoxide dismutase (SOD) and glutathione peroxidase (GPx), which scavenge reactive oxygen species (ROS) and mitigate oxidative damage to granulosa cells and oocytes^74,135 The balance between ROS production and antioxidant defenses is crucial, as excessive ROS leads to mitochondrial DNA damage, impaired meiosis, and accelerated follicular atresia. MSC transplantation has been shown to decrease ROS levels and restore redox homeostasis in ovaries affected by chemotherapy or natural aging^81,136 Moreover, MSCs activate the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway, which upregulates endogenous antioxidant defenses in ovarian cells. Activation of Nrf2 leads to increased expression of downstream antioxidant genes such as heme oxygenase-1 (HO-1), enhancing cellular resilience against oxidative insults¹³⁷ This pathway also contributes to the inhibition of ferroptosis, a form of iron-dependent cell death linked to lipid peroxidation, which has been implicated in chemotherapy-induced ovarian damage. Human umbilical cord MSCs (hUC-MSCs) suppress ferroptosis-related gene expression and fibrosis markers, thereby preserving ovarian structure and function in vitro and in vivo.¹³⁸

The paracrine effects of MSCs are pivotal in mediating these protective mechanisms. MSCs secrete a variety of cytokines, growth factors, and extracellular vesicles that modulate the ovarian microenvironment. For instance, hepatocyte growth factor (HGF) secreted by MSCs activates the PI3K-AKT signaling pathway, promoting granulosa cell proliferation and survival while inhibiting apoptosis¹³⁹ Additionally, MSC-derived exosomes inhibit the NLRP3 inflammasome-mediated pyroptosis pathway, reducing inflammatory cell death and oxidative stress in granulosa cells, which further supports follicular integrity and ovarian function⁷⁶ The anti-inflammatory properties of MSCs complement their antioxidative actions, collectively contributing to the restoration of hormonal balance and folliculogenesis.

Recent studies have also highlighted the role of MSC-derived apoptotic vesicles (apoVs) in ovarian protection. These vesicles deliver regulatory proteins such as RNF43, which inhibit aberrant WNT/β-catenin signaling in theca cells, a pathway implicated in follicular dysfunction and ovarian aging. By modulating WNT signaling, apoVs help maintain follicle homeostasis and improve oocyte quality, thereby enhancing fertility outcomes in models of polycystic ovary syndrome (PCOS) and aging¹⁴⁰ This novel mechanism underscores the multifaceted roles of MSCs and their secreted components in combating ovarian senescence.

Furthermore, MSC therapy has been shown to improve mitochondrial function in ovarian cells, which is essential for energy production and redox balance. Enhanced mitochondrial biogenesis and reduced mitochondrial DNA damage following MSC transplantation contribute to improved oocyte quality and follicular development^74,141 The restoration of mitochondrial integrity is closely linked to the activation of antioxidant pathways and suppression of apoptosis, forming an integrated network of protective effects.

In summary, MSCs exert anti-apoptotic and anti-oxidative stress effects through multiple interconnected mechanisms: upregulation of the Bcl-2/Bax ratio to inhibit mitochondrial apoptosis; secretion of antioxidant enzymes to neutralize ROS; activation of the Nrf2/ARE pathway to enhance endogenous antioxidant defenses; inhibition of ferroptosis and inflammasome-mediated pyroptosis; modulation of WNT signaling via apoptotic vesicles; and improvement of mitochondrial function. These actions collectively preserve granulosa cell and oocyte viability, promote follicular development, restore hormonal balance, and ultimately improve ovarian function and fertility in premature ovarian insufficiency and ovarian aging models. The paracrine secretion of bioactive factors and extracellular vesicles by MSCs is central to these therapeutic effects, offering promising avenues for cell-based and cell-free regenerative therapies for ovarian insufficiency.^{64,74,76,133,135,138–140}

Challenges and Risks of Stem Cell Therapy for Primary Ovarian Insufficiency

Tumorigenicity and Safety Issues

Stem cell therapies for premature ovarian insufficiency (POI) have garnered significant attention due to their regenerative potential; however, concerns regarding tumorigenicity and safety remain paramount, especially when considering different stem cell types such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs). ESCs and iPSCs possess intrinsic pluripotency and unlimited proliferative capacity, which, while advantageous for differentiation into various cell types, also confer a risk of teratoma or other tumor formation post-transplantation. The unlimited self-renewal potential of these pluripotent stem cells necessitates rigorous screening and monitoring protocols to mitigate the risk of aberrant growths such as teratomas. For instance, studies have demonstrated that iPSCs can differentiate into functional ovarian cells and restore fertility in animal models of chemotherapy-induced ovarian failure; however, the risk of tumorigenesis remains a critical barrier to clinical translation^64,142 The differentiation protocols and purification steps must be optimized to ensure the elimination of undifferentiated cells that could contribute to tumor formation.

In contrast, MSCs, derived from sources such as bone marrow, adipose tissue, umbilical cord, and menstrual blood, exhibit a markedly lower tumorigenic potential due to their multipotent nature and limited proliferative capacity. MSCs have been widely studied in POI models, showing promising results in restoring ovarian function through paracrine effects, immunomodulation, and promotion of folliculogenesis without evidence of tumor formation^35,143 Nonetheless, long-term in vitro expansion of MSCs can induce chromosomal abnormalities and senescence, which may increase the risk of malignant transformation. Therefore, establishing standardized quality control systems, including karyotype analysis and senescence markers, is essential to ensure the safety of MSC preparations intended for clinical use.^144,145

Another critical safety consideration is the immunogenicity and potential for immune rejection, particularly in allogeneic transplantation settings. While MSCs are generally considered to have low immunogenicity and possess immunosuppressive properties, reducing the likelihood of rejection, some patients have experienced adverse reactions such as fever and allergic responses following MSC transplantation^30,103 The immunomodulatory effects of MSCs, including the promotion of regulatory T cells and secretion of anti-inflammatory cytokines, contribute to their therapeutic efficacy but also necessitate careful monitoring to avoid unintended immunosuppression or infection risk^78,128 In contrast, ESCs and iPSCs, especially when not fully differentiated, may elicit stronger immune responses, underscoring the importance of immune compatibility assessments and possibly the use of autologous or HLA-matched cells to minimize rejection.^146,147

Preclinical studies have also highlighted organ-specific toxicities associated with stem cell transplantation. For example, high-dose intraovarian injection of human umbilical cord MSCs (hUC-MSCs) in rats led to inflammatory cell infiltration in the ovaries and oviducts, with increased mortality at doses approximately 50 times the standard clinical dose, indicating a narrow therapeutic window and the need for dose optimization¹⁰³ Such findings emphasize the necessity for comprehensive toxicological evaluations, including acute and chronic toxicity, immunotoxicity, and histopathological assessments, before clinical application.

Furthermore, the paracrine mechanisms by which MSCs exert therapeutic effects—through secretion of extracellular vesicles (EVs), cytokines, and growth factors—offer a potential avenue to circumvent some safety concerns associated with live cell transplantation. MSC-derived EVs have demonstrated efficacy in restoring ovarian function and fertility in POI models without the risks of uncontrolled cell proliferation or immune rejection^148,149 This cell-free therapeutic strategy may enhance safety profiles, although standardization of EV isolation, characterization, and dosing remains a challenge.

In summary, while ESCs and iPSCs offer unparalleled differentiation potential, their tumorigenic risks necessitate stringent control measures and cautious clinical translation. MSCs present a safer alternative with lower tumorigenicity and immunogenicity, yet require standardized quality control to prevent chromosomal abnormalities and adverse immune reactions. The development of cell-free therapies using MSC-derived EVs may further improve safety. Overall, addressing tumorigenicity and immunological safety is critical to advancing stem cell therapies for POI from bench to bedside.

Stem Cell Homing Efficiency and Survival Rate

The therapeutic efficacy of stem cell transplantation for premature ovarian insufficiency (POI) is critically influenced by the homing efficiency and survival rate of the transplanted cells within the ovarian microenvironment. Intravenous administration of mesenchymal stem cells (MSCs), the most commonly used stem cell type in regenerative therapies for POI, faces significant challenges due to the low proportion of cells that successfully migrate and engraft into the target ovarian tissue. Studies have consistently demonstrated that following systemic infusion, a majority of MSCs are sequestered in non-target organs such as the lungs, liver, and spleen, with less than 1% of the administered cells homing to the ovaries^35,150 This limited homing efficiency substantially restricts the regenerative potential of MSCs in restoring ovarian function.

The ovarian microenvironment in POI is characterized by ischemia, inflammation, and oxidative stress, which further compromise the survival of transplanted stem cells. The hostile milieu leads to rapid apoptosis and clearance of MSCs post-transplantation, with survival rates often reported below 10%^29,151 To overcome these obstacles, various preconditioning strategies have been explored to enhance MSC viability and homing capacity. Hypoxic preconditioning, for instance, has been shown to upregulate chemokine receptors such as CXCR4 on MSCs, thereby improving their responsiveness to stromal cell-derived factor-1 (SDF-1) gradients expressed in injured ovarian tissue^150,152 Similarly, genetic modifications to overexpress homing-related molecules or anti-apoptotic genes have demonstrated improved engraftment and prolonged survival in preclinical models.^99,153

In addition to cellular preconditioning, the use of biomaterial scaffolds has emerged as a promising approach to enhance stem cell retention and survival within the ovary. Hydrogels and collagen-based matrices provide a three-dimensional physical support that mimics the extracellular matrix, offering protection from mechanical stress and facilitating nutrient diffusion^37,123 For example, GelMA hydrogel microspheres encapsulating MSC-derived exosomes have been developed to achieve sustained release and prolonged retention in ovarian tissue, resulting in enhanced therapeutic outcomes such as reduced granulosa cell apoptosis and improved follicular development¹²³ Similarly, collagen scaffolds have been employed to localize MSCs within the ovary, mitigating their rapid clearance and promoting paracrine signaling essential for tissue repair.¹⁵⁴

The route of administration also significantly impacts homing efficiency and cell survival. Direct intraovarian injection ensures a higher local concentration of stem cells, bypassing systemic sequestration and enhancing therapeutic efficacy, as evidenced by increased follicle counts and hormone restoration in animal models^93,104 However, concerns regarding invasiveness and potential tissue damage limit its clinical application. Conversely, intravenous infusion is less invasive but suffers from poor ovarian targeting, necessitating adjunctive strategies such as low-intensity pulsed ultrasound (LIPUS) to stimulate chemokine receptor expression and improve MSC migration to the ovary.¹⁵²

Moreover, the inflammatory and hypoxic conditions within the POI ovary not only reduce stem cell survival but also modulate their functional properties. MSCs exhibit a dual nature under these stressors, where inflammation can suppress granulosa cell estradiol secretion and increase apoptosis, yet MSCs can counteract these effects through paracrine secretion of cytokines and growth factors¹⁵¹ Preconditioning MSCs under hypoxia enhances their antioxidant capacity and secretion of pro-angiogenic factors, further supporting ovarian tissue regeneration.³⁷

In summary, the limited homing efficiency and low survival rate of MSCs after systemic administration remain major barriers to effective stem cell therapy for POI. Strategies to enhance homing, such as chemokine receptor upregulation, preconditioning, genetic modification, and the use of biomaterial scaffolds, have shown promise in preclinical studies. Biomaterial-based delivery systems not only improve cell retention but also provide a supportive niche that mitigates the hostile ovarian microenvironment. Optimizing the route of administration and combining these approaches may synergistically improve the therapeutic outcomes of stem cell transplantation in POI. Future research should focus on translating these strategies into clinical protocols, ensuring safety, efficacy, and reproducibility to realize the full potential of stem cell therapy for ovarian regeneration.

Ethical and Regulatory Issues

The ethical and regulatory landscape surrounding stem cell therapy for premature ovarian insufficiency (POI) is complex and multifaceted, reflecting the diverse origins and characteristics of the stem cells involved. Embryonic stem cells (ESCs), derived from early-stage embryos, inherently involve the destruction of embryos, which raises significant ethical concerns. This has led many countries to impose strict regulations or outright bans on the clinical use of ESCs, limiting their application despite their potent pluripotency and therapeutic promise. The ethical debate centers on the moral status of the embryo and the balance between potential medical benefits and respect for embryonic life, which continues to influence policy and public acceptance globally.¹⁵⁵

In contrast, induced pluripotent stem cells (iPSCs) circumvent many of these ethical issues as they are generated by reprogramming adult somatic cells back to a pluripotent state without the need for embryos. This technology offers a promising alternative for regenerative medicine, including ovarian regeneration, by providing patient-specific cells that reduce immunogenicity risks. However, the reprogramming process itself is not without concerns. It may introduce genetic mutations or epigenetic abnormalities that could compromise the safety and efficacy of iPSC-derived therapies. Long-term safety data remain limited, necessitating rigorous preclinical and clinical evaluation to assess tumorigenicity, genomic stability, and functional integration before widespread clinical adoption.^156,157

Regulatory frameworks for stem cell therapies vary considerably across countries, reflecting differing ethical standards, scientific assessments, and healthcare policies. Clinical trials involving stem cells must adhere to Good Clinical Practice (GCP) standards to ensure patient safety, informed consent, and data integrity. This includes comprehensive protocols for patient selection, stem cell sourcing, manufacturing under Good Manufacturing Practice (GMP) conditions, and post-treatment monitoring. For example, clinical studies using mesenchymal stem cells (MSCs) for POI have demonstrated promising outcomes but also highlight the need for standardized protocols and long-term follow-up to monitor adverse events and therapeutic durability.^23,24

Moreover, the administration routes and dosages of stem cells require careful regulatory scrutiny. Preclinical toxicity studies, such as those evaluating human umbilical cord mesenchymal stem cells (hUC-MSCs) injected intraovarianly in animal models, have identified potential target organ toxicities and dose-dependent mortality, underscoring the importance of defining maximum tolerated doses and safety margins before clinical translation¹⁰³ Similarly, intravenous administration of amniotic mesenchymal stem cells (hAMSCs) has been evaluated for safety and efficacy, with preclinical data supporting their biocompatibility and therapeutic potential at defined doses.¹⁰⁷

Informed consent is a critical ethical component, ensuring that patients understand the experimental nature of stem cell therapies, potential risks, benefits, and alternatives. Given the novelty and complexity of these treatments, clear communication and ethical oversight are essential to protect vulnerable patient populations, particularly women with infertility seeking hope through innovative interventions. Long-term follow-up is also mandated to detect delayed adverse effects such as tumor formation or immune reactions, which may not be apparent in short-term studies.¹⁵⁸

Finally, the heterogeneity of stem cell populations and the variability in their biological behavior pose challenges for regulatory agencies. Advances in engineering mesenchymal stem cells and optimizing their therapeutic properties raise additional regulatory considerations regarding genetic modifications and the use of exosomes or secretomes as cell-free therapies. These innovations require updated guidelines to address safety, efficacy, and manufacturing consistency.^99,159

In summary, while stem cell therapy for POI holds great promise, ethical concerns—particularly regarding embryonic stem cells—and safety issues related to iPSC reprogramming necessitate cautious progression. Regulatory policies must balance innovation with patient protection, requiring adherence to GCP, rigorous preclinical safety assessments, standardized clinical protocols, and robust informed consent processes. Multinational harmonization of regulations and ongoing ethical discourse will be vital to facilitate the responsible clinical translation of stem cell therapies in reproductive medicine.

Research on Stem Cells Combined with Other Treatment Strategies

Stem Cell Combined with Hormone Replacement Therapy

Hormone replacement therapy (HRT) has long been the cornerstone treatment for premature ovarian insufficiency (POI), primarily aimed at alleviating hypoestrogenic symptoms and improving quality of life by restoring hormonal balance. However, HRT alone does not restore ovarian function such as folliculogenesis, ovulation, or fertility. Recent advances in regenerative medicine have introduced stem cell therapy as a promising approach to repair and regenerate damaged ovarian tissue, potentially restoring ovarian reserve and function. Combining stem cell therapy with HRT may offer synergistic benefits by improving the ovarian microenvironment, thereby enhancing stem cell survival, engraftment, and therapeutic efficacy.

HRT can modulate the ovarian microenvironment by replenishing estrogen and other sex steroids, which are critical for maintaining follicular health and stromal support. This hormonal milieu creates a more favorable niche for transplanted stem cells, such as bone marrow-derived mesenchymal stem cells (BMSCs), to exert their regenerative effects. Animal studies have demonstrated that the combination of HRT and BMSC transplantation leads to superior recovery of ovarian function compared to either treatment alone. For instance, in chemotherapy-induced POI rodent models, co-administration of HRT with BMSCs resulted in increased follicle counts, improved estrous cyclicity, and normalized serum hormone levels, surpassing the outcomes observed with monotherapy. These findings suggest that HRT not only alleviates systemic hypoestrogenism but also enhances the local ovarian environment, promoting stem cell-mediated repair mechanisms including anti-apoptosis, angiogenesis, and immunomodulation.

Despite these promising preclinical results, the long-term risks associated with HRT, such as increased incidence of breast cancer and cardiovascular disease, remain a significant concern, especially with prolonged use. Therefore, the integration of stem cell therapy with HRT necessitates careful consideration of patient selection, dosing regimens, and duration of hormone administration to maximize benefits while minimizing adverse effects. Moreover, the timing of HRT initiation relative to stem cell transplantation may be critical; early hormonal support could prime the ovarian niche for stem cell engraftment, whereas delayed administration might limit therapeutic synergy.

In clinical contexts, HRT remains indispensable for managing menopausal symptoms and preventing osteoporosis in POI patients, but it falls short in restoring fertility. Stem cell therapies, particularly those utilizing mesenchymal stem cells from various sources such as bone marrow, adipose tissue, and umbilical cord, have shown potential in preclinical and early clinical studies to regenerate ovarian tissue and improve reproductive outcomes. The combination approach leverages the symptomatic relief provided by HRT and the regenerative capacity of stem cells, aiming for a more comprehensive treatment strategy.

Future research should focus on optimizing protocols for combined therapy, including identifying the most effective stem cell types, delivery methods, and HRT regimens. Additionally, long-term follow-up studies are essential to evaluate the safety and efficacy of combined treatments, particularly concerning oncogenic risks associated with hormone therapy. Advances in biomaterials and engineered scaffolds may further enhance stem cell survival and function within the hormonally modulated ovarian microenvironment.

In summary, the integration of stem cell therapy with hormone replacement therapy represents a promising frontier in the treatment of premature ovarian insufficiency. By improving the ovarian microenvironment through hormonal modulation, HRT can potentiate the reparative effects of stem cells, leading to enhanced restoration of ovarian function. However, balancing therapeutic efficacy with safety concerns remains paramount, necessitating rigorous clinical evaluation and individualized treatment planning. This combined approach holds the potential to not only alleviate menopausal symptoms but also to restore fertility and improve the overall reproductive health of women affected by POI.^2,25,124,160

Stem Cells Combined with Biomaterial Scaffolds

The integration of stem cells with biomaterial scaffolds represents a promising advancement in the treatment of premature ovarian insufficiency (POI) or premature ovarian insufficiency (POI), aiming to enhance stem cell retention, viability, and therapeutic efficacy within the ovarian microenvironment. Biomaterials such as gelatin sponges and hyaluronic acid (HA) hydrogels serve as three-dimensional (3D) carriers that provide a supportive matrix for stem cells, facilitating cell adhesion, proliferation, and differentiation, which are critical for effective ovarian tissue regeneration. These scaffolds mimic the natural extracellular matrix (ECM), offering a conducive milieu that preserves stem cell functionality and promotes paracrine signaling essential for ovarian repair.

In preclinical models, particularly in POI rat models induced by chemotherapeutic agents like cyclophosphamide, the transplantation of umbilical cord mesenchymal stem cells (UC-MSCs) loaded onto collagen-based scaffolds has demonstrated superior outcomes compared to direct stem cell injection. The collagen scaffold prolongs the retention time of UC-MSCs within the ovarian tissue, thereby enhancing their reparative effects. This is evidenced by significant improvements in follicular development, restoration of estrous cycles, and normalization of sex hormone levels, including estradiol and anti-Müllerian hormone (AMH), which are indicative of ovarian reserve recovery. Histological analyses reveal increased numbers of healthy follicles and reduced granulosa cell apoptosis in scaffold-assisted stem cell therapy groups, underscoring the scaffold’s role in creating a protective niche that mitigates the hostile post-chemotherapy ovarian environment.^31,139

Moreover, the use of autocrosslinked hyaluronic acid (HA) hydrogels as a biomaterial scaffold has been shown to not only extend the survival and retention of UC-MSCs in the ovary but also to enhance their secretory function. HA scaffolds facilitate the activation of key signaling pathways such as PI3K-AKT via paracrine mechanisms, promoting follicular survival and growth. Hepatocyte growth factor (HGF), identified as a pivotal cytokine secreted by UC-MSCs within this scaffold environment, mediates these regenerative effects by stimulating angiogenesis and inhibiting apoptosis in ovarian cells. The combination of UC-MSCs with HA scaffolds thus represents a synergistic approach that improves the therapeutic efficiency of stem cell transplantation in both physiological aging and chemically induced POI models.¹³⁹

Innovations in biomaterials have also introduced smart, stimuli-responsive scaffolds such as thermosensitive hydrogels, which enable controlled and sustained release of stem cells or their derivatives. These intelligent materials respond to environmental cues like temperature changes, allowing minimally invasive delivery and localized retention of stem cells within the ovary. Controlled release systems improve treatment precision by maintaining optimal cell concentrations at the target site over extended periods, reducing systemic dispersion and enhancing regenerative outcomes. Although specific studies on thermosensitive hydrogels in POI models are emerging, their potential to fine-tune stem cell delivery and improve ovarian tissue regeneration is promising and warrants further investigation.¹³⁹

In addition to enhancing cell retention and viability, biomaterial scaffolds contribute to the modulation of the ovarian microenvironment by supporting extracellular matrix remodeling, reducing fibrosis, and attenuating oxidative stress and inflammation. These effects collectively create a more favorable niche for endogenous folliculogenesis and ovarian function restoration. The paracrine factors secreted by UC-MSCs within scaffolds, including growth factors, cytokines, and extracellular vesicles, orchestrate these reparative processes by activating anti-apoptotic pathways, promoting angiogenesis, and regulating immune responses. For instance, activation of the PI3K-AKT signaling pathway has been consistently implicated in mediating the protective and regenerative effects of stem cell therapy combined with biomaterial scaffolds.^43,139

Clinical translation of stem cell-biomaterial scaffold combinations is supported by preliminary human studies demonstrating improved ovarian reserve markers and fertility outcomes following UC-MSC transplantation. The use of biomaterial scaffolds may address current limitations such as low cell engraftment rates and transient therapeutic effects observed with direct stem cell injections. By providing a 3D microenvironment that mimics native ovarian ECM, scaffolds enhance stem cell survival and function, potentially leading to more durable and effective treatments for POI patients.^102,161

In summary, the combination of stem cells with biomaterial scaffolds such as gelatin sponges, collagen matrices, and hyaluronic acid hydrogels offers a multifaceted strategy to improve the therapeutic efficacy of stem cell transplantation for premature ovarian insufficiency. These scaffolds provide structural support, enhance cell retention and viability, and facilitate controlled release, thereby promoting follicular regeneration and hormonal recovery. The incorporation of smart, responsive biomaterials further refines delivery precision and treatment outcomes. Future research should focus on optimizing scaffold composition, understanding the molecular mechanisms underlying scaffold-stem cell interactions, and conducting rigorous clinical trials to validate safety and efficacy in human POI patients.

Stem Cell Combined with Gene Editing Technology

Evidence status: predominantly preclinical and developmental.

Gene editing should currently be regarded as an experimental strategy rather than an established therapeutic approach for POI. The integration of stem cell therapy with gene editing technologies, particularly CRISPR/Cas9, represents a cutting-edge approach to enhance the therapeutic potential for premature ovarian insufficiency (POI) or premature ovarian insufficiency (POI). By genetically modifying stem cells to overexpress survival-promoting genes such as Bcl-2 and HIF-1α, or anti-inflammatory factors like IL-10, researchers aim to augment the intrinsic regenerative and immunomodulatory capabilities of mesenchymal stem cells (MSCs). For instance, studies have demonstrated that MSCs expressing heme oxygenase-1 (HO-1) can activate the JNK/Bcl-2 signaling pathway, thereby enhancing granulosa cell viability and reducing apoptosis, which are critical for ovarian function restoration¹²⁶ Similarly, overexpression of anti-apoptotic genes like Bcl-2 in MSCs can improve follicular survival and hormone secretion, as evidenced by increased estradiol and anti-Müllerian hormone (AMH) levels in treated animal models⁶¹ The anti-inflammatory cytokine IL-10, which can be upregulated through gene editing, plays a pivotal role in modulating immune responses and promoting ovarian tissue repair, as shown in placental MSC transplantation studies where IL-10 elevation correlated with suppression of NLRP3 inflammasome-mediated pyroptosis.¹²⁸

Gene editing also offers the possibility of correcting mutations in genes directly implicated in POI pathogenesis, such as FMR1 and BMP15. Mutations in FMR1 are associated with fragile X-associated premature ovarian insufficiency, and BMP15 mutations disrupt folliculogenesis. Although direct gene correction in ovarian cells remains challenging, editing stem cells prior to transplantation could circumvent these obstacles by providing a source of genetically corrected cells capable of restoring ovarian function. However, the complexity of gene editing in stem cells includes concerns about off-target effects, which may lead to unintended genetic alterations and potential tumorigenicity. For example, the use of CRISPR/Cas9 requires precise targeting to avoid genomic instability, and current delivery methods must be optimized to ensure safety and efficacy.³⁰

The combined strategy of stem cell therapy and gene editing necessitates a careful balance between maximizing therapeutic efficacy and minimizing risks. While gene-modified MSCs can exhibit enhanced survival, paracrine secretion, and immunomodulation, the risk of oncogenic transformation due to insertional mutagenesis or off-target gene editing remains a significant safety concern. Preclinical studies have emphasized the importance of rigorous screening for genomic integrity and tumorigenic potential before clinical application¹⁰⁴ Moreover, the immunogenicity of genetically modified cells must be evaluated, as alterations in surface antigens or secreted factors could provoke immune rejection or adverse inflammatory responses.¹⁶²

In summary, the application of CRISPR/Cas9-mediated gene editing to modify stem cells holds great promise for enhancing the treatment of POI by overexpressing survival and anti-inflammatory genes or correcting pathogenic mutations. Nonetheless, the technical challenges of precise gene editing, potential off-target effects, and safety concerns such as tumorigenicity must be thoroughly addressed. Future research should focus on developing safer gene editing platforms, improving delivery systems, and establishing standardized protocols for evaluating the efficacy and safety of gene-edited stem cells. This integrated approach could ultimately lead to more effective and durable therapies for restoring ovarian function while ensuring patient safety.

Emerging and Translational Research Priorities

The approaches discussed in this section should be interpreted as research priorities rather than established clinical strategies. Their feasibility and clinical value remain to be determined in appropriately controlled studies.

Optimization and Standardization of Stem Cell Sources

The optimization and standardization of stem cell sources are critical for advancing stem cell-based therapies for premature ovarian insufficiency (POI) and related ovarian dysfunctions. A major focus has been the development of non-invasive and easily accessible stem cell sources, such as urine-derived stem cells (USCs) and menstrual blood-derived stem cells (MenSCs), which offer significant advantages over traditional sources by minimizing ethical concerns and invasive procedures. USCs, isolated from urine, have demonstrated multipotent differentiation capabilities and express typical mesenchymal stem cell markers, including CD29, CD44, CD73, and CD90, while lacking hematopoietic markers such as CD34 and CD45. Notably, a subpopulation of USCs expressing CD133 exhibits enhanced chondrogenic differentiation potential, which may translate into improved regenerative efficacy when their exosomes are applied therapeutically. For example, exosomes derived from CD133+ USCs have been shown to promote bone-tendon interface healing more effectively than those from unsorted USCs, likely due to their superior ability to induce differentiation of bone marrow mesenchymal stem cells (BMSCs) into chondroblasts¹⁶³ Similarly, MenSCs have emerged as a promising source due to their accessibility and robust secretory profile. Exosomes derived from MenSCs have been demonstrated to stimulate chondrocyte extracellular matrix synthesis, enhancing collagen type II and TGF-β3 receptor expression, which are crucial for tissue regeneration and repair¹⁶⁴ These findings underscore the therapeutic potential of non-invasively obtained stem cells and their derivatives in regenerative medicine, including ovarian repair.

To ensure reproducibility and clinical applicability, establishing stem cell banks is essential for the standardized production, storage, and quality control of allogeneic stem cell products. Stem cell banks facilitate the generation of consistent cell batches with defined phenotypic and functional characteristics, thereby reducing variability between preparations. This standardization is particularly important given the heterogeneity inherent in stem cell populations and the influence of donor variability, culture conditions, and passage number on cell properties. Quality control measures include phenotypic characterization by flow cytometry, assessment of differentiation potential, and evaluation of secretome profiles, including extracellular vesicle (EV) content. The implementation of good manufacturing practice (GMP) guidelines in stem cell processing ensures sterility, safety, and efficacy, which are prerequisites for clinical translation¹⁶⁵ Moreover, the establishment of stem cell banks enables the ready availability of therapeutic cells or their derivatives, facilitating timely treatment interventions.

In parallel with cellular therapies, the exploration of stem cell-derived extracellular vesicles, particularly exosomes, as cell-free therapeutic agents has gained considerable momentum. Exosomes are nanosized vesicles (30–150 nm) that mediate intercellular communication by transferring bioactive molecules such as proteins, lipids, mRNAs, and microRNAs. They recapitulate many of the therapeutic effects of their parent stem cells but circumvent several limitations associated with cell transplantation, including risks of tumorigenicity, immune rejection, and embolism formation. For instance, mesenchymal stem cell (MSC)-derived exosomes have demonstrated immunomodulatory, anti-apoptotic, and pro-angiogenic effects in various preclinical models, including ovarian insufficiency, cartilage repair, and nerve regeneration^148,166,167 The use of exosomes also offers advantages in terms of storage stability, ease of administration, and potential for engineering to enhance targeting and therapeutic payload delivery¹⁶⁸ Importantly, exosome-based therapies reduce the ethical and safety concerns associated with stem cell transplantation, making them attractive candidates for clinical application.

Recent advances have highlighted the therapeutic efficacy of exosomes derived from differentiated stem cell progeny, such as granulosa-like cells (GLCs), which may provide enhanced tissue specificity and functional benefits. For example, extracellular vesicles from differentiated GLCs have shown superior ovarian tropism and efficacy in restoring ovarian function in chemotherapy-induced POI rat models compared to undifferentiated umbilical cord MSC-derived EVs. Mechanistically, these GLC-EVs promote primordial follicle activation via pathways involving PLAU-mediated phosphorylation of FOXO3A, underscoring the potential for precision cell-free therapies tailored to ovarian regeneration¹⁶⁹ Furthermore, the developmentof biomaterial-based delivery systems, such as hydrogels incorporating exosomes from urine-derived stem cell subpopulations, has demonstrated enhanced localized and sustained release, promoting tissue repair in spinal cord injury models¹⁷⁰ Such delivery platforms may be adapted for ovarian applications to improve therapeutic outcomes.

Despite these promising developments, challenges remain in the isolation, characterization, and standardization of exosome preparations. Variability in isolation methods, purity, and cargo composition can affect therapeutic efficacy and reproducibility. Advanced characterization techniques, including nanoparticle tracking analysis, transmission electron microscopy, and proteomic profiling, are essential to ensure quality control. Moreover, the establishment of reference standards and GMP-compliant production protocols is critical for clinical translation^171,172 Addressing these challenges will facilitate the development of reliable, safe, and effective exosome-based therapies for POI and other regenerative indications.

In summary, the optimization and standardization of stem cell sources, including the development of non-invasive cell sources like USCs and MenSCs, the establishment of stem cell banks for consistent production, and the exploration of exosome-based cell-free therapies, represent pivotal advances in regenerative medicine. These strategies collectively aim to enhance therapeutic efficacy, safety, and accessibility, thereby accelerating the translation of stem cell-derived products into clinical treatments for premature ovarian insufficiency and related disorders.

Individualized Treatment Plan Formulation

Premature ovarian insufficiency (POI), also known as premature ovarian insufficiency (POI), is a heterogeneous condition with multifactorial etiologies including genetic abnormalities, autoimmune disorders, and iatrogenic causes such as chemotherapy or surgery. The complexity of its pathogenesis necessitates a personalized approach to stem cell therapy, tailoring treatment based on the underlying cause, patient age, and ovarian reserve status to optimize outcomes. Selecting the most appropriate type of mesenchymal stem cells (MSCs) and administration protocols is critical for maximizing therapeutic efficacy and safety.

Genetic causes of POI often involve chromosomal abnormalities or mutations affecting DNA repair pathways, leading to accelerated follicular depletion through apoptosis or other cell death mechanisms. In such cases, stem cells with robust regenerative and anti-apoptotic capacities, such as human umbilical cord-derived MSCs (hUC-MSCs) or human amniotic epithelial cells (hAECs), may be preferred due to their demonstrated abilities to restore ovarian reserve and improve folliculogenesis by modulating DNA damage responses and promoting granulosa cell survival^29,37 For autoimmune-related POI, characterized by immune-mediated ovarian damage and elevated pro-inflammatory cytokines, MSCs with potent immunomodulatory properties, such as bone marrow-derived MSCs (hBMSCs), may be more suitable These cells can suppress inflammation, inhibit pyroptosis in granulosa cells, and restore hormonal balance, as evidenced by studies showing improved estrous cycles and follicle counts following hBMSC transplantation in autoimmune POI models^30,173 Iatrogenic POI, often induced by chemotherapy agents like cyclophosphamide, requires MSCs capable of counteracting oxidative stress, ferroptosis, and mitochondrial dysfunction. Umbilical cord MSCs and MSC-derived exosomes have shown efficacy in protecting granulosa cells from chemotherapy-induced ferroptosis and enhancing mitochondrial biogenesis, thereby preserving ovarian function.^33,174

Patient age and ovarian reserve status, assessed by biomarkers such as anti-Müllerian hormone (AMH) levels and antral follicle count (AFC), are pivotal in guiding stem cell therapy. Younger patients with higher residual ovarian reserve tend to respond better to MSC transplantation, achieving improved follicular development and even successful pregnancies, as demonstrated in clinical trials involving UCMSC ovarian injections^93,102 Conversely, older patients or those with severely diminished ovarian reserve may require adjunctive strategies or combination therapies to enhance stem cell efficacy. For instance, co-administration of MSCs with agents like ginsenoside Rg1 or melatonin has been shown to potentiate anti-apoptotic effects and antioxidant capacity, thereby improving ovarian recovery in chemotherapy-induced POI models.^161,175

Biomarkers play a crucial role in predicting treatment response and enabling precision medicine in POI. AMH and AFC are established indicators of ovarian reserve, with higher baseline levels correlating with better outcomes post-MSC therapy^93,176 Additionally, gene expression profiling of ovarian tissue and granulosa cells can provide insights into the molecular milieu, including activation of pathways such as PI3K/AKT/mTOR and PTEN/FOXO3, which are involved in follicular activation and survival. Monitoring these molecular signatures before and after treatment can help tailor MSC selection and dosing regimens^162,177 Emerging evidence also supports the use of inflammatory markers and immune cell profiles to stratify patients with autoimmune POI and optimize immunomodulatory stem cell therapies.^173,178

The integration of artificial intelligence (AI) and big data analytics offers promising avenues to refine individualized treatment plans. By analyzing large datasets encompassing patient demographics, genetic information, biomarker levels, and clinical outcomes, AI algorithms can identify patterns predictive of therapeutic success and adverse events. This approach facilitates optimization of MSC dosage, administration frequency, and combination strategies. For example, studies have highlighted an inverted U-shaped dose-response relationship for MSCs in POI treatment, suggesting that both insufficient and excessive cell doses may compromise efficacy^179,180 AI-driven models can assist in determining the optimal therapeutic window for each patient. Moreover, combining MSC therapy with other modalities such as extracellular vesicles, pharmacological agents, or tissue engineering scaffolds can be systematically evaluated through computational simulations to enhance ovarian regeneration.^181,182

In summary, the formulation of individualized treatment plans for POI using stem cell therapy requires a comprehensive assessment of disease etiology, patient age, ovarian reserve, and biomarker profiles. Selecting the appropriate MSC source—be it umbilical cord, bone marrow, amniotic membrane, or pluripotent stem cell-derived MSCs—and tailoring administration protocols are essential for maximizing therapeutic benefits. The incorporation of AI and big data analytics holds great potential to optimize dosing, timing, and combination therapies, thereby advancing precision medicine in the management of POI. Continued research and clinical trials are warranted to validate these strategies and establish standardized guidelines for personalized stem cell-based interventions in premature ovarian insufficiency.

Long-Term Safety and Efficacy Evaluation

The long-term safety and efficacy of stem cell therapy for premature ovarian insufficiency (POI) or premature ovarian insufficiency (POI) remain critical concerns that must be addressed through rigorous clinical investigation. To establish the therapeutic value of stem cell interventions, large-scale, multicenter, randomized controlled clinical trials with extended follow-up periods of at least 5 to 10 years are essential. Such trials would provide robust data on the durability of ovarian function restoration, reproductive outcomes including fertility and live birth rates, and the incidence of adverse events over time. Current preclinical and early clinical studies have demonstrated promising short-term improvements in ovarian hormone levels, folliculogenesis, and fertility restoration following transplantation of various mesenchymal stem cells (MSCs) derived from sources such as human umbilical cord, bone marrow, amnion, and adipose tissue^{4,43,47,158,183} However, these studies often involve small sample sizes, limited controls, and short observation windows, limiting the ability to assess long-term safety and sustained efficacy.

A key aspect of long-term safety evaluation is the monitoring for rare but serious adverse events such as tumorigenesis and autoimmune reactions. Although MSCs generally exhibit low immunogenicity and tumorigenic potential, concerns remain regarding their paracrine effects and potential to induce aberrant tissue growth or immune dysregulation^103,152 To this end, establishing comprehensive patient registries and real-world data collection systems is vital. These registries would enable systematic surveillance of treated patients, capturing data on ovarian function, reproductive outcomes, and any late-onset complications including neoplasms or autoimmune diseases. For example, a toxicity study in rats receiving intraovarian injections of human umbilical cord MSCs revealed inflammatory infiltration in ovaries and oviducts at high doses, with mortality linked to doses approximately 50 times the clinical standard, underscoring the importance of dose optimization and safety monitoring.¹⁰³

In addition to clinical and registry data, the development and application of non-invasive monitoring technologies are crucial for real-time tracking of stem cell fate, engraftment, and therapeutic effects. Techniques such as circulating extracellular vesicle (exosome) analysis, including profiling of microRNAs and proteins, offer promising biomarkers for monitoring ovarian response and stem cell activity post-transplantation^47,99,183 Advanced imaging modalities, including ultrasound-guided injections and molecular imaging, can facilitate precise delivery and localization of stem cells or their derivatives, as well as longitudinal assessment of ovarian morphology and vascularization^184,185 For instance, ultrasound-guided intraovarian injection of human umbilical cord MSC-derived exosomes demonstrated improved ovarian hormone levels, follicular development, and fertility outcomes in animal models, highlighting the utility of imaging in both therapeutic administration and monitoring.¹⁸⁵

Furthermore, the heterogeneity of MSC populations and their secreted extracellular vesicles necessitates careful characterization to ensure consistent therapeutic efficacy and safety. Clonal MSC populations and defined extracellular vesicle subpopulations have shown differential regenerative capacities and immunomodulatory effects in preclinical POI models, suggesting that selecting specific subpopulations may optimize outcomes and reduce risks^99,132 Genetic and epigenetic modifications of MSCs, such as conservative hypomethylation, have also been explored to enhance their therapeutic potential while maintaining safety profiles.¹⁸⁶

Long-term follow-up studies in patients who have undergone hematopoietic stem cell transplantation (HSCT) provide valuable insights into ovarian function recovery and fertility potential post-treatment. Data indicate that spontaneous menarche and pregnancies can occur years after HSCT, although premature ovarian insufficiency remains common, emphasizing the need for fertility preservation strategies and long-term monitoring in this population^187–195 Similarly, clinical trials investigating adipose-derived MSC therapy for female sexual dysfunction and ovarian insufficiency are underway, with designs incorporating safety endpoints and functional assessments over extended periods¹⁸⁹, 197–199.

In conclusion, the long-term safety and efficacy evaluation of stem cell therapy for POI/POI requires a multifaceted approach integrating large-scale randomized trials, patient registries, advanced non-invasive monitoring techniques, and molecular characterization of therapeutic cell populations. Addressing these aspects will be pivotal to translating promising preclinical findings into safe, effective, and standardized clinical treatments that restore ovarian function and fertility while minimizing risks.

Stem cell-based interventions represent an active area of experimental research in premature ovarian insufficiency (POI), with the strongest evidence currently derived from preclinical models. MSCs, particularly bone marrow- and umbilical cord-derived cells, have repeatedly produced improvements in ovarian histology, endocrine parameters, follicular measures, and reproductive performance in animal models. These effects are most consistently associated with paracrine signaling, immunomodulation, anti-apoptotic and antioxidant activity, angiogenesis, and remodeling of the ovarian microenvironment. Direct differentiation into functional ovarian cells remains less firmly established and should not be assumed to be the principal mechanism.

Human evidence is considerably more limited. Small, predominantly non-randomized or uncontrolled studies have reported menstrual recovery and changes in AMH, FSH, estradiol, AFC, or ovarian morphology, but these surrogate outcomes do not establish restoration of the primordial follicle pool or fertility. Pregnancy reports are also difficult to attribute solely to stem-cell treatment because POI may involve intermittent ovarian activity and because IVF, embryo transfer, donor oocytes, or other concomitant interventions may contribute to conception. Current clinical evidence therefore supports feasibility and potential biological activity rather than established clinical effectiveness.

The field also faces substantial translational challenges, including variability in cell source and potency, dose and administration route, manufacturing and quality control, uncertain durability of cell effects, and limited long-term safety data. Biomaterials, extracellular vesicles, and gene-editing approaches may address selected limitations, but most remain preclinical or early translational strategies. These approaches should not be described as ovarian regeneration or restoration of ovarian reserve without direct evidence supporting those claims.

Future clinical development should prioritize standardized cell characterization, transparent reporting of dose and route, appropriate control groups, clinically meaningful reproductive endpoints, and sufficiently long follow-up. Particular attention should be given to live birth and other reproductive outcomes alongside standardized ovarian-function measures. Multicenter controlled studies will be needed to determine whether the biological signals observed in early studies translate into reproducible patient benefit.

Overall, stem cell therapy should currently be considered an investigational strategy for POI. The distinction between improved ovarian function, altered ovarian reserve markers, and demonstrated fertility is essential for accurate interpretation of the rapidly evolving evidence. A cautious, evidence-based approach will be necessary to define which stem-cell products, patients, routes, and treatment regimens, if any, can ultimately provide clinically meaningful benefit.

Funding Statement

This work was supported by Open Research Projects of the Qinghai Province High-Altitude Medical Laboratory(2026-KF-01) and Qinghai Province obstetrics and gynecology disease clinical medical research center(2024-SF-L03).

Disclosure

Shuna Zhang and Fei Wang contributed equally as co-first authors. The authors report no conflicts of interest in this work.