The mechanism and protective strategies of follicle injury after ovarian tissue cryopreservation and thawed transplantation: a review

Abstract

The incidence of early-onset malignancies in reproductive-aged women is rising, necessitating effective fertility preservation strategies. Ovarian tissue cryopreservation (OTC) remains the sole option for prepubertal girls and patients requiring urgent oncologic treatment. However, post-transplant follicular attrition—driven by ischemia-reperfusion injury, oxidative stress, and aberrant primordial follicle activation—remains a major barrier. This comprehensive review elucidates the multiscale mechanisms of cryopreservation-induced follicular damage and evaluates cutting-edge strategies, including antifreeze protein-engineered cryoprotectants, angiogenesis-modulating scaffolds, and mTOR pathway inhibition. By integrating recent advances in biomaterial science and cryobiology, this study provides actionable insights to enhance OTC clinical efficacy and advance reproductive medicine.

Background

In recent years, the rising incidence of malignant tumors with increasingly younger onset poses a significant public health threat; while advances in treatment have improved long-term survival rates [1], achieving remission or tumor-free status has made conception a pressing concern for many young survivors. Common antitumor therapies—including radiotherapy, chemotherapy, and targeted therapy—can severely impair gonadal function, leading to infertility or permanent reproductive loss [2], necessitating fertility preservation as a critical challenge in reproductive medicine. Clinically available methods include embryo cryopreservation, oocyte cryopreservation, ovarian tissue cryopreservation (OTC), in vitro maturation, and gonadotropin-releasing hormone agonists. Notably, OTC is distinguished by its lack of age or timing restrictions, serving as the sole option for prepubertal girls and patients unable to delay antitumor therapy initiation [3, 4]. Though proposed for conditions like Turner syndrome, its clinical application remains debated due to concerns about follicular depletion patterns and graft viability [5, 6].

OTC involves surgically obtaining the ovarian cortex, sectioning it into slices (∼1 mm thick × 5–10 mm long/wide), vitrifying or slow-freezing the tissue, and storing it in liquid nitrogen; when needed, thawed tissue is transplanted back [7]. Since the first live birth via OTC in 2004, over 300 successful births have validated its efficacy [8–10], leading the American Society for Reproductive Medicine (ASRM) to reclassify it as a standard (non-experimental) therapy in 2019 [11].

Beyond restoring fertility, transplantation also recovers endocrine function [9, 12, 13]. However, significant challenges persist, including potential contamination by malignant cells increasing recurrence risk [3, 7, 14–16], and follicular depletion/damage from freeze-thaw-transplantation processes shortening graft lifespan [9, 17, 18]. As graft longevity relies directly on surviving primordial follicles [19, 20], the 50–90% follicular loss post-transplantation underscores the need to investigate injury mechanisms and develop protective strategies [9, 20, 21].

This review analyzes mechanisms underlying ischemia/hypoxia-induced injury and aberrant primordial follicle activation post-transplant, while evaluating strategies to enhance OTC efficacy via antioxidants, cytokines, stem cells and novel biomaterials—offering novel insights into follicular injury and advancing OTC treatment.

Mechanism of follicle injury in OTC and thawed ovarian tissue transplantation

Cryodamage in OTC

Slow freezing remains the conventional cryopreservation technique in clinical practice, accounting for the majority of reported live births following ovarian tissue transplantation. This method involves specimen immersion in low-concentration cryoprotectants, followed by controlled cooling via computerized programmable freezers. Tissues are progressively frozen at defined rates (− 35 °C to − 130 °C) prior to long-term storage in liquid nitrogen [22]. Notwithstanding its technical maturity, slow freezing exhibits inherent limitations: (1) heterogeneous cooling rates between cortical and medullary compartments; (2) prolonged isothermal phases due to latent heat release during phase transition; (3) mechanical damage from extracellular ice crystallization; and (4) cryoprotectant toxicity risks [23]. Histopathological and molecular analyses of cryopreserved ovarian tissue from transgender individuals confirm significant cryodamage versus fresh controls, including diminished follicular morphological integrity, elevated apoptosis indices, and downregulation of gap junction proteins—though subsequent in vitro culture confirms retained follicular functionality [24]. Complementary studies further demonstrate 42% depletion of primordial follicle reserves and substantial stromal cell viability loss (< 65%) post-slow-freezing [25].

In contrast, vitrification employs ultra-rapid cooling of tissues in high-concentration cryoprotectants to achieve a glassy, ice crystal-free state [22]. While this technique offers operational simplicity, rapid cooling rates, and reduced equipment dependency, it introduces risks of cryoprotectant-induced chemical toxicity, osmotic shock, and devitrification (ice nucleation upon temperature fluctuations exceeding the glass transition point) [26].

Comparative studies on vitrification and slow freezing methods for OTC have become the focus of recent research. Wang et al. demonstrated that although both slow freezing and vitrification downregulated the mRNA levels of ZP3 and CYP11A, vitrification had a more pronounced negative impact on the development and morphological normality of human primordial follicles, as well as on the mRNA expression of anti-Müllerian hormone (AMH), compared to slow freezing [27]. However, other studies have indicated that vitrification, in contrast to slow freezing, reduces apoptosis and DNA damage while improving follicular morphological normality, follicular density, and stromal cell integrity [25, 28]. Conversely, research by Klocke et al. and three meta-analyses revealed no significant differences in follicular morphological normality, estradiol secretion after in vitro culture, follicular proliferation, or apoptosis rates between slow freezing and vitrification [25, 29–31]. In 2023, Chung et al. conducted xenotransplantation analyses of human ovarian tissues following slow freezing and vitrification and found that both methods effectively maintained the survival of primordial follicles and were clinically applicable [32]. Although slow freezing retains its status as the conventional gold standard, vitrification has achieved significant breakthroughs, with recent global live births demonstrating preliminary feasibility [33–36]. Current consensus indicates slow freezing as preferable for large tissue fragments, while vitrification requires optimization for cryoprotectant toxicity mitigation (e.g., cell strainer carriers) and standardization of protocols; future directions include biomaterial-enhanced transplantation and large-scale comparative trials to resolve ongoing controversies.

Hypoxia, ischemia-reperfusion injury (IRI) and cell death

During the early post-transplantation phase, hypoxic-ischemic injury and IRI constitute major contributors to follicular depletion in thawed ovarian grafts [20, 37]. Due to the absence of vascular anastomosis, revascularization is essential for restoring adequate oxygen and nutrient supply to the transplanted tissue [38]. Prior to neovascular establishment, oxygen diffusion from surrounding tissues proves insufficient to meet metabolic demands [19], with human ovarian grafts enduring approximately 5 days of hypoxia [20, 39–41]. This hypoxic milieu disrupts mitochondrial membrane potential homeostasis, induces endoplasmic reticulum stress, and activates apoptotic cascades, thereby amplifying follicular and stromal cell apoptosis. Concurrently, adenosine triphosphate (ATP) depletion triggers organelle and membrane degradation, generating reactive oxygen species (ROS) and inflammatory mediators that exacerbate cellular damage [9]. Granulosa and stromal cells—exhibiting higher proliferative and metabolic rates than oocytes—demonstrate heightened susceptibility to post-transplantation apoptosis, suggesting their extensive loss may partially account for follicular depletion in grafts [42].

Revascularization timelines differ across species, requiring 7 days in murine models versus 10 days in humans for functional vessel formation and oxygen partial pressure stabilization [39, 43]. Subsequent blood supply restoration paradoxically generates substantial ROS, which induces vascular endothelial dysfunction, increases microvascular permeability, promotes tissue edema, and amplifies inflammatory responses, collectively aggravating cellular injury and apoptosis—a hallmark of IRI [44, 45]. Notably, pyroptosis has emerged as a key mechanism in myocardial and renal IRI; similarly, elevated expression of pyroptosis-related proteins (caspase-1, NLRP3) occurs in autografted mouse ovaries, with significantly higher levels in thawed versus fresh grafts [46], indicating its potential role in post-transplantation follicular injury.

Autophagy, a critical homeostatic process for primordial follicle maintenance under physiological conditions, is dysregulated following transplantation: while genetic ablation of autophagy-related genes accelerates primordial follicle loss [47], excessive autophagic flux under ischemic stress triggers cellular dysfunction. Rapid activation of autophagy occurs post-transplantation—predominantly in granulosa and stromal cells of growing follicles (both pre-existing and newly activated primary follicles)—with intensity correlating positively with ischemia severity [20]. Intriguingly, moderate autophagy may mitigate follicular loss by facilitating revascularization (via endothelial cell autophagy enhancement) and ROS/damaged organelle clearance; conversely, severe hypoxia-induced hyperactivation can drive follicular depletion [48].

Aberrant activation of primordial follicles

Primordial follicles residing in the ovarian cortex are maintained in a quiescent state by the unique biophysical microenvironment—characterized by high collagen density, compact tissue architecture, and limited vascularization—which shields follicles from mechanical stimuli that could induce premature activation, thereby constituting a critical determinant of ovarian reserve [39]. During OTC, however, tissues are susceptible to physical, solute, osmotic, and cryogenic injuries that may trigger aberrant primordial follicle activation or death. Comparative analyses reveal significantly elevated proportions of developing follicles in both fresh and cryopreserved transplanted tissues versus non-transplanted controls, indicating that cryopreservation and transplantation procedures accelerate primordial follicle activation, leading to substantial depletion of the follicular reserve and compromised graft longevity [49]. Mechanistically, Celik et al. demonstrated that vitrification-thawing upregulates phosphorylated ribosomal protein S6 kinase (p-s6K) and activates the mTOR pathway—a master regulator of primordial follicle activation—resulting in abnormal follicular recruitment [50]. Complementary studies confirm that the PI3K/PTEN/AKT signaling pathway, when activated during tissue processing phases (transportation, slow freezing, in vitro culture), similarly promotes primordial follicle activation [51]. Furthermore, cryopreservation-induced ROS overproduction activates multiple pathways (MAPK, JAK/STAT, PI3K/AKT/mTOR, NF-κB), potentially exacerbating PI3K/PTEN/AKT-driven follicular recruitment [52]. Besides, cortical tissue sectioning during OTC also disrupts structural integrity, dysregulating the Hippo signaling pathway: reduced phosphorylation of Yes-associated protein (YAP) enables nuclear translocation of non-phosphorylated YAP, driving aberrant primordial follicle activation [53, 54]. Synergistic crosstalk between Hippo and PI3K/AKT pathways may further amplify this effect [53]. Lastly, the absence of developing follicles in grafts reduces AMH production—a key paracrine inhibitor of primordial follicle initiation—contributing to extensive post-transplantation activation [55].

Ovarian fibrosis

Ovarian fibrosis—characterized by aberrant fibroblast proliferation and excessive extracellular matrix (ECM) deposition—constitutes a pivotal pathological driver of diminished ovarian reserve [56]. Key histopathological hallmarks include ovarian capsular thickening, pathological collagen accumulation, stromal hyperplasia, and extensive follicular atresia, collectively impairing folliculogenesis, ovulation competence, and reproductive outcomes [57]. Clinically, fibrotic ovaries demonstrate attenuated responsiveness to assisted reproductive technologies and elevated infertility risks, profoundly compromising female reproductive health [56, 58]. This fibrotic transformation can be triggered by multifactorial insults including surgical trauma, chronic inflammation, reproductive aging, or immune dysregulation involving macrophage-mediated cytokine cascades (e.g., TGF-β) and dysregulated wound healing pathways, thereby accelerating maladaptive ECM remodeling [59]. Critically, in OTC, freeze-thaw cycles and IRI exacerbate cortical fibrosis, disrupting the stromal niche essential for maintaining primordial follicle quiescence and follicular development [60]. Mechanistically, ECM-derived biomechanical signals transduced via integrin receptors and cytoskeletal networks modulate nuclear gene expression and chromatin architecture, perpetuating fibrotic phenotypes through sustained positive feedback loops [61]. Nevertheless, the spatiotemporal dynamics of post-transplantation fibrotic remodeling remain incompletely characterized, necessitating further investigation into microenvironmental regulators.

Strategies to enhance the efficiency of OTC

To prolong graft longevity and enhance the efficiency of OTC, various strategies have been employed to mitigate follicular loss, including antioxidants, pro-angiogenic agents, primordial follicle activation inhibitors, and novel cryoprotectants. New biomaterials, including hydrogels and decellularized scaffolds, are also gradually attracting researchers’ interest (Table 1).

Table 1.

Damage mechanisms and protective strategies in OTC​ and thawed transplantation

Damage Type​​	​​Specific Mechanisms​​	​​Protective Strategies​
​​Cryodamage​	• Mechanical ice crystal injury (slow freezing) • Cryoprotectant toxicity (vitrification) • Follicle integrity decline • Stromal cell viability < 65%	• ​​Novel cryoprotectants​​: - AFPs: Suppress ice growth - GO: Matches ice prism plane O-O spacing - Zwitterionic polymers: Mitigate osmotic stress via charge shielding • ​​Biomaterial carriers​​: - Alginate hydrogels: Mimic ECM microenvironment
​Ischemia-Reperfusion Injury (IRI)​	• Hypoxia (> 5 days) inducing mitochondrial dysfunction • ROS burst triggering apoptosis • Pyroptosis-related protein upregulation (caspase-1, NLRP3)	• ​​Antioxidants​​: - Melatonin: Activates Nrf2/ARE pathway, increase SOD/GSH activity - NAC: Inhibits TNF-α/caspase-3, increase follicle survival rate - EPO: Promotes neovascularization and reduces oxidative DNA damage - Vitamin C/E/Trolox: Inhibit lipid peroxidation cascade - Allopurinol: Blocking superoxide anion production - Resveratrol: Activates SIRT1, decrease apoptosis • ​​Pro-angiogenic agents​​: - bFGF/VEGF with or without hydrogels: increase microvascular density - MSCs: Secrete VEGF/Ang-1 - Ang2: Mediates angiogenesis and vascular remodeling - FSH/hMG: Stimulate angiogenesis and reduce granulosa and stromal cell apoptosis
Aberrant Primordial Follicle Activation	• mTOR/p-S6K signaling activation • Hippo pathway dysregulation • Insufficient AMH secretion	• ​​mTOR inhibitors​​: - Rapamycin: Inhibits p-S6K, inhibit aberrant primordial follicle activation • ​​AMH replacement​​: - Recombinant AMH: Preserves primordial follicle pool - AMH-overexpressing endothelial cells: Decrease follicular atresia
​​Ovarian Fibrosis​	• TGF-β-mediated ECM overdeposition • Abnormal integrin-cytoskeleton signaling	• ​​Anti-fibrotic biomaterials​​: - Decellularized scaffolds: Preserve ECM structure, decrease collagen fragments - Melatonin + endothelial cell hydrogels: Inhibit fibrosis via NO pathway - Ang2 + VEGF: suppression of TGF-β/Smad signaling pathway

Abbreviation: AFPs: antifreeze proteins; GO, graphene oxide; ECM, extracellular matrix; SOD: superoxide dismutase; GSH: glutathione; NAC: N-acetylcysteine; EPO: erythropoietin; IRI: ischemia-reperfusion injury; ROS: reactive oxygen species; bFGF: basic fibroblast growth factor; VEGF: vascular endothelial growth factor; MSCs: mesenchymal stem cells; Ang2: angiopoietin-2; FSH: follicle-stimulating hormone; hMG: human menopausal gonadotropin; AMH: anti-Müllerian hormone

Antioxidants

Melatonin

Melatonin exerts potent direct free radical scavenging capacity and indirectly enhances endogenous antioxidant defenses through receptor-mediated pathways, thereby conferring comprehensive antioxidative, anti-apoptotic, and anti-inflammatory protection during ovarian tissue cryopreservation and transplantation [62, 63]. Mechanistically, melatonin directly neutralizes ROS and reactive nitrogen species, while upregulating glutathione (GSH), glutathione peroxidase, superoxide dismutase (SOD), and catalase activities via activation of the Nrf2/ARE signaling pathway—significantly reducing oxidative biomarkers (malondialdehyde, nitric oxide) in vitrified-thawed ovaries and improving follicular morphological preservation [64]. ​​Notably​​, melatonin attenuates cryopreservation-induced apoptosis by upregulating heat shock protein 90 expression and suppressing caspase-3 activation in rat ovarian tissues [65], while murine studies confirm its capacity to mitigate oxidative stress and follicular apoptosis post-vitrification [66].

Beyond cryoprotection, melatonin enhances transplant efficacy: in murine autotransplantation models, intraperitoneal administration accelerates estrous cycle recovery, elevates progesterone and estradiol levels, increases ovarian volume, and preserves multi-stage follicle counts by suppressing ROS burst and inflammatory cytokine release [67]. ​Furthermore​​, melatonin promotes primordial follicle activation and cumulus-oocyte complex integrity in grafts, correlating with elevated proportions of developing follicles [68]. ​​Critically​​, co-delivery of melatonin with CD₁₄₄ endothelial cells in alginate-fibrin hydrogels synergistically stimulates graft revascularization and reduces fibrotic remodeling in heterotopically transplanted vitrified-thawed ovaries, demonstrating its dual role in enhancing vascularization and mitigating stromal damage [69].

N-acetylcysteine (NAC)

NAC functions as a metabolic precursor for intracellular cysteine and GSH biosynthesis, conferring potent antioxidant activity through direct free radical scavenging and indirect enhancement of endogenous redox defenses [39, 70]. In murine models of ovarian autotransplantation, NAC administration significantly improved follicular survival and developmental competence within grafts by mitigating oxidative stress-induced apoptosis [71]. Synergistic strategies combining NAC with estradiol further demonstrated enhanced cytoprotective effects on follicular integrity and steroidogenesis in rodent ovarian grafts, suggesting combinatorial therapeutic potential [72]. Notably, xenotransplantation studies utilizing cryopreserved-thawed human ovarian tissue revealed that NAC attenuates IRI via dual antioxidant and anti-inflammatory mechanisms—specifically suppressing ROS generation, TNF-α signaling, and caspase-3 activation—thereby increasing primordial follicle survival rates by 2.3-fold compared to untreated controls [73].

Others

Beyond melatonin and NAC, diverse antioxidants have been investigated for OTC and transplantation. Erythropoietin​​ (EPO) promotes neovascularization via vascular endothelial growth factor (VEGF) upregulation and reduces oxidative DNA damage in rodent/canine models, preserving stromal integrity and restoring follicular function [74–79]; Vitamin E​​ and its water-soluble analog ​​Trolox​​ inhibit lipid peroxidation cascade; pre-transplant intraperitoneal vitamin C/E injection reduced MDA in rat autografts, while Trolox supplementation prevented endoplasmic reticulum-derived cytoplasmic vacuolization during primate ovarian cryopreservation [80, 81]; Allopurinol​​, a xanthine oxidase inhibitor, attenuates IRI by blocking superoxide anion production, enhancing follicular survival in murine transplantation models [82]; Resveratrol​​ activates SIRT1-mediated antioxidant pathways (SOD2, CAT) and inhibits NF-κB-driven inflammation, reducing apoptosis and improving follicular density in vitrified-warmed mouse ovaries [40].

Angiogenesis and anti-fibrosis

Basic fibroblast growth factor (bFGF) and VEGF

bFGF and VEGF are widely utilized in cryopreserved ovarian tissue transplantation, either as monotherapies or in combinatorial strategies, to mitigate ischemic injury and enhance graft functionality. To address the rapid enzymatic degradation of free bFGF in vivo, studies have employed fibrin hydrogel scaffolds for its sustained local delivery during co-transplantation with fresh ovarian tissue. ​​Notably​​, bFGF encapsulation significantly improved primordial follicle survival rates, stimulated neoangiogenesis (evidenced by increased microvessel density), and enhanced stromal cell proliferation in murine allografts [83]. Furthermore​​, dual loading of bFGF and VEGF into fibrin hydrogels synergistically promoted follicular preservation, accelerated revascularization (CD31⁺ endothelial cell recruitment), and restored estrous cyclicity in fresh murine ovarian grafts, surpassing the efficacy of single-factor treatments [84]. In a xenotransplantation model​, pretreatment of vitrified-warmed ovarian tissue with bFGF-supplemented culture media prior to xenotransplantation enhanced graft vascularization, increased follicular survival (quantified by reduced caspase-3 activity), and stimulated cellular proliferation (Ki-67⁺ cell fraction) in SCID mouse models [85, 86]. Critically​​, combined peri-transplantation administration—entailing ex vivo pretreatment with bFGF/VEGF followed by subcutaneous factor delivery at the graft site—significantly augmented angiogenesis while suppressing apoptosis (TUNEL⁺ cell reduction) in fresh human ovarian xenografts, with synergistic effects observed in rabbit models [87].

Mesenchymal stem cells (MSCs)

MSCs, as multipotent adult stem cells, are isolatable from diverse tissues including bone marrow, umbilical cord, menstrual blood, endometrium, and adipose tissue [88]. Adipose-derived MSCs (ASCs) demonstrate dual proangiogenic and anti-apoptotic capacities owing to their high accessibility and paracrine activity. ASCs promote angiogenesis via secretion of VEGF and angiopoietin-1, alongside direct differentiation into endothelial lineage cells [47, 89, 90]. A “two-step” transplantation strategy has been proposed for human OTC xenotransplantation: ASCs pre-loaded onto fibrin scaffolds are implanted at the graft site 14 days prior to ovarian tissue transplantation, significantly enhancing neovascularization and attenuating IRI in grafts [89, 91, 92]. Beyond revascularization, ASC-secreted cytokines (e.g., HGF, IGF-1) directly reduce follicular apoptosis and suppress primordial follicle activation by downregulating PI3K/AKT and Hippo signaling pathways, thereby preserving the dormant follicle pool [47, 91]. ASCs further contribute to long-term ovarian reserve maintenance by sustaining physiological follicular distribution across developmental stages [55].

Other MSC types—including bone marrow-derived MSCs (BM-MSCs), visceral peritoneal MSCs, and human umbilical cord MSCs (HucMSCs)—also exert protective effects in ovarian transplantation. Encapsulation of cryopreserved-thawed human ovarian tissue with BM-MSCs in Matrigel upregulates pro-angiogenic factors (VEGF, FGF2, angiogenin), enhancing revascularization while reducing primordial follicle apoptosis and structural damage [93]. In murine autotransplantation models, both BM-MSCs and peritoneal MSCs restored endocrine function and accelerated estrous cycle recovery [94]. Hypoxia-preconditioned HucMSCs further amplify these benefits through HIF1α/VEGFA pathway activation, significantly suppressing apoptosis and promoting follicular survival in human ovarian grafts [95].

Significantly, MSC-derived extracellular vesicles (MSC-EVs)—comprising exosomes (50–150 nm), microvesicles (100–1000 nm), and apoptotic bodies (500–5000 nm)—retain therapeutic functionalities akin to parental cells while exhibiting superior stability and reduced immunogenicity [88]. Accumulating evidence supports MSC-EV efficacy in ovarian insufficiency: rodent models demonstrate that MSC-EVs stimulate granulosa cell proliferation, inhibit apoptosis via miRNA-mediated anti-apoptotic gene regulation (e.g., Bcl-2 upregulation, Bax suppression), and restore endocrine function, thereby offering a novel cell-free strategy to enhance cryopreserved ovarian transplantation outcomes [96–98].

Biomaterials

Hydrogels—soft biocompatible biomaterials with three-dimensional hydrophilic crosslinked networks—effectively confine ice crystal propagation within their matrices by creating a constrained aqueous microenvironment during cryopreservation [99]. Cell encapsulation in hydrogels significantly minimizes permeable cryoprotectant agent (pCPA) concentrations while maintaining vitrification efficacy, thereby reducing CPA-associated cytotoxicity [100, 101]. Calcium ion-crosslinked sodium alginate hydrogels, widely utilized in tissue engineering, replicate an ECM-mimetic niche that enhances porosity and mechanical resilience. This architecture promotes encapsulated cell survival and proliferation, positioning alginate hydrogels as promising cryoprotective platforms [102–104]. Notably​​, integrating alginate with complementary biomaterials (e.g., fibrin, polycaprolactone, hyaluronic acid, chitosan, or RGD peptides) synergistically optimizes composite physicochemical properties. Such modifications overcome inherent limitations of pure alginate systems and establish a favorable microenvironment for in vitro follicle culture, significantly enhancing follicular growth and developmental competence [69, 105–109]. Henry et al. demonstrated that collagen matrices loaded with VEGF accelerated vascular ingrowth in thawed sheep ovarian xenografts, achieving detectable perfusion within 3 days and significantly increasing microvessel density (> 1.8-fold vs. controls) in immunodeficient mice [110]. Shikanov et al. utilized VEGF₁₆₈/heparin-binding peptide hydrogels for mouse ovary autotransplantation, observing functional vascularization within 21 days and subsequent natural conception with live births [111]. Chung et al. engineered platelet-derived growth factor (PDGF)-encapsulated fibrin hydrogels that enhanced neovascularization, reduced DNA damage , improved ovulation rates, and elevated embryo development in transplantation models—highlighting their potential to mitigate IRI [112]. Furthermore​​, incorporating bioactive molecules (e.g., epigallocatechin gallate) into hydrogel matrices not only attenuates oxidative stress but also activates angiogenic pathways (e.g., VEGF, HIF-1α), creating a pro-regenerative microenvironment for post-thaw follicular survival and functional recovery [108]. These findings underscore the potential of hydrogel-based delivery systems in advancing the cryopreservation of ovarian tissues for reproductive medicine applications.

Decellularized scaffolds—generated via physical/chemical/enzymatic removal of cellular components—preserve tissue-specific ECM components, structural integrity, and biomechanical cues while eliminating immunogenicity [113, 114]. These scaffolds provide a tissue-specific, immune-privileged microenvironment that enhances graft-host integration and endogenous repair mechanisms: Eivazkhani et al. validated that NaOH-mediated decellularization of human/ovine ovaries better preserved ECM ultrastructure and follicular reconstitution capacity than sodium dodecyl sulfate-treated scaffolds, attributable to reduced collagen fragmentation [115]. Kutluk et al. achieved the first successful pregnancy following minimally invasive transplantation of cryopreserved ovarian tissue using human decellularized ECM scaffolds combined with robotic surgery, demonstrating stable ovarian function over 24 months—a milestone in clinical translation [116]. Despite these advances​​, challenges persist in recellularization efficiency and scalability. While three dimension-printed ovarian scaffolds show promise for in vitro follicle maturation [117], their application in transplantation remains nascent, necessitating further optimization of bioink composition and mechanical stability.

Others

Angiopoietin-2 (Ang2) directly mediates angiogenesis and vascular remodeling, exhibiting prominent expression at sites of active neovascularization. Under ischemic/hypoxic conditions, Ang2 initiates neovessel formation by destabilizing existing vasculature to facilitate endothelial sprouting. In murine models of vitrified-warmed ovarian autotransplantation, Ang2 supplementation increased the proportion of morphologically intact follicles to 73.1% (vs. 48.7% in controls) by enhancing graft neovascularization [118]. Critically​​, combined delivery of Ang2 and VEGF synergistically amplified vascular reconstruction in xenotransplanted vitrified-warmed bovine ovarian tissues, preserving normal follicular architecture (65.9% morphological integrity vs. 55.1% in untreated grafts), reducing follicular apoptosis by 9.3%, and attenuating fibrotic remodeling [119].

Follicle-stimulating hormone (FSH) confers cytoprotection in vitrified-warmed ovarian transplantation models through dual mechanisms: (1) upregulating gap junction proteins [connexin 43 (Gja1) and connexin 37 (Gja4)] to enhance cell-cell communication within follicular units, and (2) activating VEGF/VEGFR2 signaling to stimulate angiogenesis. These actions collectively reduce granulosa and stromal cell apoptosis and preserve follicular integrity [120]. Furthermore​​, preconditioning freshly isolated murine ovarian tissue with FSH-containing medium prior to transplantation amplified pro-angiogenic effects, elevating VEGF and bFGF expression by 26.27-fold and 33.2-fold, respectively, thereby accelerating functional revascularization [121]. Consistently​​, human menopausal gonadotropin (hMG) pretreatment in murine autotransplantation models demonstrated conserved gonadotropin-mediated vascular support, improving graft survival rates by 38% and underscoring the therapeutic potential of gonadotropin priming in clinical ovarian transplantation protocols [122].

Suppression of primordial follicle activation

AMH

AMH acts as a potent suppressor of primordial follicle recruitment. Recent murine studies demonstrate that recombinant AMH administration significantly reduces primordial follicle depletion in vitrified-warmed ovarian autotransplantation models by modulating key regulators within grafts—including tuberous sclerosis complex 1 (TSC1), growth differentiation factor 9 (GDF9), and p-S6K—thereby preserving > 40% of the primordial follicle pool compared to untreated controls [123]. Notably​​, while human xenograft models utilizing AMH infusion pumps in nude mice showed no significant reduction in total primordial follicle loss, AMH effectively suppressed abnormal follicular activation and progression to secondary stages by downregulating PI3K/AKT/mTOR signaling [124]. Critically​​, a synergistic strategy co-transplanting AMH-overexpressing endothelial cells with cryopreserved human ovarian tissue demonstrated dual mechanisms: enhanced graft vascularization through endothelial cell activity coupled with AMH-mediated suppression of aberrant primordial follicle activation. This combined approach preserved the primordial follicle pool and extended graft functional longevity [125].

mTOR inhibitor

Rapamycin, a potent mTOR inhibitor acting downstream of the PI3K/AKT signaling pathway, effectively suppresses aberrant primordial follicle activation during OTC. Pretreatment of murine ovaries with rapamycin prior to vitrification significantly inhibits post-transplantation follicular hyperactivation while preserving normal developmental competence, indicating a transient but critical regulatory role in maintaining follicular quiescence [126]. Notably​​, this protective effect was replicated when rapamycin was incorporated into the cryopreservation medium during programmed freezing of murine ovaries [127]. ​​Similarly​​, in vitro cultures of cryopreserved-thawed murine ovarian tissue demonstrated significant reduction in aberrant follicle activation when treated with rapamycin during the freezing protocol, attributed to suppression of PI3K/Akt/mTOR-mediated follicular recruitment [51]. ​​Critically​​, human xenotransplantation models corroborated these findings: systemic rapamycin administration in nude mouse recipients attenuated primordial follicle activation in vitrified-warmed human ovarian grafts [128].

Antifreeze proteins and engineered cryoprotectants

Antifreeze proteins (AFPs)

Natural organisms have evolved specialized adaptations to produce antifreeze proteins (AFPs), enabling survival in subzero environments by mitigating cryoinjury. AFPs employ dual cryoprotective mechanisms—thermal hysteresis and ice recrystallization inhibition—to precisely regulate ice crystal dynamics. These mechanisms constrain ice crystal growth to an average diameter ≤ 20 μm (below the threshold for organelle damage), remodel ice morphology into low-stress hexagonal prisms, and disperse ice crystals uniformly within extracellular spaces, thereby preventing localized compression on follicles while circumventing the cytotoxicity associated with conventional cryoprotectants [129, 130]. Experimental evidence​​ supports that AFPs supplementation significantly enhances post-vitrification sperm motility and mitochondrial membrane potential, improves survival rates and developmental competence of in vivo-matured mouse oocytes, reduces spindle/chromosomal aberrations , and increases cryopreserved embryo viability [131–133]. ​​In OTC models​​, AFPs reduce apoptotic follicle ratios to 12.6% (vs. 22.6% in untreated grafts), maintain follicular ultrastructural integrity, and suppress primordial follicle activation via PI3K/Akt pathway inhibition in murine systems [134–136].

Despite their potential​​, natural AFPs face scalability limitations due to complex purification processes. ​​To address this​​, Qi et al. designed a 20-residue de novo antifreeze peptide (AVD) wherein Thr⁶-Asn⁸ spacing precisely matches the oxygen lattice spacing on ice prism planes, achieving ice crystal area suppression comparable to natural AFPs. At -80 °C, AVD exhibits bifunctional activity: (1) suppressing ice crystal growth via surface adsorption, and (2) stabilizing voltage-dependent calcium channels to prevent membrane destabilization. Experimental validation demonstrated 95.7% recovery in cryopreserved lung cancer cells (3.1-fold higher than controls) and 34.9% recovery in immune cells (14.5-fold increase), with preserved mitochondrial ultrastructure and proliferative capacity equivalent to fresh cells [137]. ​​However​​, high production costs currently limit widespread application. ​​Critically​​, AFP-based cryopreservation strategies remain at the preclinical stage for human ovarian tissues, necessitating further optimization of delivery systems and validation in human-derived tissue models before clinical translation.

Novel cryoprotectants

Recent advances in AFPs research have catalyzed the development of novel cryoprotectants, with graphene oxide (GO) emerging as a promising nanomaterial for ice-crystal regulation. GO’s hexagonal carbon lattice—functionalized with hydroxyl, epoxy, and carboxyl groups—enables structural mimicry of natural AFPs by aligning its oxygen atoms with ice crystal planes, thereby adsorbing to ice surfaces and restricting crystal growth via hydrogen-bond restructuring [138]. Experimental evidence​​ demonstrates that GO supplementation during equine sperm cryopreservation significantly enhances post-thaw motility from 24.3% to 71.3% and preserves membrane integrity by reducing lipid peroxidation [139]. Notably​​, co-transplantation of GO/poly-L-lactic acid (GO/PLLA) composite nanofiber scaffolds with ovarian cortex grafts in premature ovarian insufficiency mouse models accelerated host-graft integration through nitric oxide-mediated angiogenesis, increased primordial follicle counts, and restored bilateral ovarian function via phosphorylation of endothelial nitric oxide synthase (p-eNOS) [140]. Despite this transformative potential​​, GO-based cryopreservation faces dual challenges. First, the synthesis of nanomaterials requires precise control of temperature, solvent, and other parameters, as deviations may lead to failed production. Second, their safety remains controversial, as needle-like morphologies can damage cell membranes and induce oxidative stress. Thus​​, future innovation must prioritize defect-engineered GO synthesis and surface functionalization (e.g., PEGylation) to mitigate cytotoxicity, alongside rigorous evaluation in human-derived tissue models to bridge translational gaps.

Polymer-based cryoprotectants, distinguished by high molecular weight, low cytotoxicity, and enhanced biocompatibility, represent promising alternatives to conventional agents such as dimethyl sulfoxide (DMSO). Capitalizing on mechanistic insights from AFPs, researchers have engineered diverse macromolecular architectures—including synthetic polymers, low-molecular-weight carbohydrates, and zwitterionic polyampholytes—for gamete and embryo cryopreservation. Pioneering work by Eniade et al. first identified ice recrystallization inhibition activity in small-molecule carbohydrates, demonstrating their capacity to suppress pathological ice growth through hydrogen-bond disruption at ice crystal interfaces [141]. Macromolecular polymers​​ such as polyvinylpyrrolidone (PVP) exhibit broader applicability; in human sperm cryopreservation, PVP reduces DNA fragmentation by > 40% and significantly elevates cleavage rates (90.0% vs. 69.5%) and blastocyst formation (45.4% vs. 30.9%) post-thaw [142]. This cryoprotective efficacy extends to caprine oocytes, murine oocytes, and embryos, where PVP stabilizes membrane fluidity and mitochondrial function [143–145]. Similarly, polyproline improved post-thaw cell viability compared to DMSO alone. Adding 50 mmol/L low-molecular-weight polyproline to cryopreservation media replaced approximately 1.8 mol/L DMSO and glycerol, increasing the survival rate of thawed oocytes from 95.93% to 99.11% [146, 147]. Additionally​​, zwitterionic polyampholytes—featuring balanced cationic/anionic moieties on a single chain—function as low-toxicity cryoprotectants with AFP-mimetic properties. These polymers mitigate osmotic shock through charge-shielding effects and have successfully preserved mesenchymal stem cells and red blood cells [148, 149]. Despite these advances​​, the translation of novel cryoprotectants to OTC remains limited. Future studies must prioritize in vivo validation of biocompatibility, dose optimization in human ovarian tissue models, and standardized toxicity profiling to bridge preclinical innovation and clinical implementation.

Conclusion

OTC has become the only fertility protection option for girls and women before puberty who need immediate treatment for gonadal toxicity. Although it has been adopted in clinical practice, follicular depletion after transplantation remains a major obstacle, mainly driven by two mechanisms: hypoxia, IRI accompanied by oxidative stress and cell death, and excessive activation of primordial follicles, leading to a reduction in ovarian reserve. Current research strategies aim to mitigate these losses through pharmacological interventions, including antioxidants, pro-angiogenic agents, primordial follicle activation inhibitors, and novel cryoprotectants. New biomaterials, including hydrogels and decellularized scaffolds, are also gradually attracting researchers’ interest. Notably​​, while most agents exhibit monofunctional efficacy, recent evidence underscores the superior potential of combinatorial approaches: coupling antioxidants (e.g., melatonin) with mTOR inhibitors (e.g., rapamycin) synergistically reduces oxidative damage while suppressing follicular hyperactivation, and stem cell-derived paracrine factors (e.g., exosomal miRNAs) concurrently attenuate inflammation and enhance angiogenesis. Critically​​, current evidence is predominantly derived from animal models and human xenograft studies due to the scarcity of human ovarian tissue samples and ethical constraints surrounding experimental manipulation, thereby precluding robust clinical translation. ​​Consequently​​, there is an urgent need for translational clinical trials to optimize cryoprotectant formulations, validate primordial follicle activation suppression strategies (e.g., AMH/rapamycin delivery systems), and standardize metrics for post-transplantation outcomes. Future efforts should prioritize ​​three pillars​​: (1) enhancing graft survival through multi-target interventions (e.g., combining vascularization promoters with anti-fibrotic agents), (2) restoring endocrine function via steroidogenic cell preservation, and (3) maximizing live birth rates through coordinated fertility restoration protocols, ultimately addressing the reproductive autonomy and psychosocial well-being of cancer survivors.

Biographies

Yifan Chu

obtained his master’s degree from the Second Clinical College (Tongji Hospital), Tongji Medical College, Huazhong University of Science and Technology of China in 2024. He is currently a M. D. student under the supervision of Prof. Jing Yue. His research is centered on development of ovarian tissue cryopreservation and assisted reproductive technology.

Prof. Jing Yue

is currently a chief physician and the vice director at Reproductive Medicine and Genetics Centre, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, specializing in assisted reproductive technology, fertility preservation and gynecological endocrinology. She received her Ph.D. in reproductive medicine from Huazhong University of Science and Technology in 2008. Her research focuses on fertility preservation, endometrial receptivity and endometrial flora.

Author contributions

This study was designed by JY and YC. YC, JZ (Jialiang Zhang), LW, JX and JC collected and analyzed the literatures. The draft was written and corrected by YC, JZ (Jialiang Zhang), LW, JX and JC and was critically reviewed by JY, MY, RM, JZ (Juepu Zhou) and XH. JY agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the final manuscript.

Funding

This work was supported by the National Key Research & Development Program of China (grant number 2022YFC2702503), the Open Project of Hubei Clinical Research Center for Reproductive Medicine (grant number 2025RMOF005) and the Peking University Third Hospital Open Subject of the Department of Obstetrics and Gynecology (grant number BYSYSZKF2022008).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.