A Comprehensive Review of Advances in Ovarian Tissue Transplantation: From Cryopreservation Protocols to Graft Revascularization Dynamics

1. Introduction

The continuous advancement of diagnostic and therapeutic approaches in oncology has substantially improved survival rates among young and adult women diagnosed with neoplasms [1]. However, anticancer treatments frequently impair reproductive function and may result in premature ovarian failure [2]. In this context, cryopreservation combined with ovarian tissue transplantation has emerged as a promising strategy for fertility preservation and for the restoration of endocrine activity [3]. Beyond human applications, these techniques also hold considerable potential for conservation programs involving species with reproductive challenges or those threatened with extinction, thereby contributing to biodiversity preservation [4,5].

Depending on the type of recipient, ovarian tissue transplantation may be classified as autotransplantation (within the same individual), isotransplantation (between genetically identical individuals), allotransplantation (between individuals of the same species), or xenotransplantation (between individuals of different species) [6]. Xenotransplantation has been widely used in experimental studies and in reproductive research involving domestic species.

Ovarian tissue transplantation involves the reimplantation of cortical fragments into different anatomical sites. These sites may be classified as orthotopic, when the tissue is positioned near its original location, or heterotopic, such as in the subcutaneous tissue, intraperitoneal cavity, or beneath the renal capsule, all of which can support effective revascularization [7,8]. Evidence indicates that heterotopic transplantation offers practical advantages related to anesthesia and surgical procedures, thereby facilitating endocrine recovery [9]. According to Oktay & Marin [10], orthotopic ovarian transplantation results in higher gamete and embryo quality. However, the endocrine function restoration rate and longevity are similar between orthotopic and heterothopic approaches. When feasible, orthotopic should be preferred for those who intend to conceive, although a less invasive heterotopic can be performed for those who primarily desire ovarian endocrine function [10].

Despite encouraging outcomes, including more than 200 births reported worldwide, the technique still faces limitations related to ischemia and delayed revascularization, which may lead to necrosis, fibrosis, and the loss of stromal and follicular cells [11,12,13]. Ischemia begins immediately after tissue removal and is typically alleviated only around seven days after transplantation [14,15]. Consequently, selecting biologically suitable implantation sites is essential to ensure graft survival and functionality [16]. Several factors influence this process, including oxidative stress [17], hypoxia [18], hormonal and molecular interactions [19], and variations in grafting protocols [16].

The pre-transplantation phase, particularly the method of tissue preservation, plays a central role in minimizing cellular damage. Vitrification has emerged as an efficient approach to ovarian tissue cryopreservation because it reduces ice crystal formation and offers operational practicality. It has yielded favorable results in different species, including humans [20,21], small ruminants [22,23,24], felids [25,26,27], and canids [28,29,30,31,32]. However, elevated oxidative stress in vitrified samples may compromise cellular viability due to the high concentrations of cryoprotectants and the rapid cooling rates used. The incorporation of antioxidants has therefore been proposed as a strategy to mitigate these deleterious effects [33].

After vitrification, ovarian tissue can resume its physiological function either through in vivo or in vitro culture, supporting follicular development and the production of viable oocytes for fertilization [22,24,34]. Understanding the combined effects of vitrification and transplantation is thus essential for advancing reproductive biotechnologies and promoting fertility preservation. This review aims to integrate recent evidence on ovarian tissue cryopreservation and transplantation in veterinary species, with emphasis on the cellular mechanisms underlying follicular survival and activation, the factors that modulate graft revascularization, and the histological and histochemical approaches used for tissue assessment. The originality of this contribution lies in its comparative synthesis of cryopreservation methods, strategies for preserving the follicular microenvironment, the use of angiogenic modulators, and grafting protocols, providing an integrated perspective not yet consolidated in the veterinary literature.

2. Ovarian Tissue Cryopreservation

Ovarian tissue cryopreservation consists of storing cells and tissues at ultra-low temperatures (−150°C to −196°C), allowing metabolic activity to decrease and enabling long-term preservation until normal development can later resume [35]. This powerful technique has emerged as an effective strategy for maintaining ovarian function and fertility, providing protection against premature ovarian failure and other causes of follicular reserve depletion. Among its advantages are the preservation of the entire pool of primordial follicles, its applicability at any age or estrous cycle stage [36], the safeguarding of genetic material in women prior to oncologic treatments, reduced ethical concerns compared with oocyte and embryo cryopreservation [37], and the conservation of genetic material from high-value animals or endangered species [38]. Collectively, these factors underscore their broad reproductive potential and value for genetic preservation.

Cryopreservation can be performed using either slow freezing or vitrification (Figure 1), which differ primarily in cooling rate [32]. In slow freezing, temperature is gradually reduced (~1°C/min) with the use of low concentration cryoprotectants; however, ice crystal formation still occurs and may damage cellular structures. In vitrification, ice formation is essentially prevented by achieving a glass-like state through the application of high concentrations of cryoprotectants combined with rapid cooling to -196°C. This method is faster, simpler, and does not require specialized equipment [37].

Several ovarian tissue vitrification protocols employing intracellular cryoprotectants (ethylene glycol and dimethyl sulfoxide) combined with extracellular cryoprotectants (sucrose), in stepwise increasing concentrations of intracellular agents (10% and 20%), have been successfully applied in different domestic species, including sheep [39], goats [22], and cattle [40]. Among these protocols, the Ovarian Tissue Cryosystem (OTC) can be highlighted as a metallic, hermetically sealed device that prevents direct contact between the tissue and liquid nitrogen, thereby avoiding contamination. Additionally, the system allows the vitrification solution to be removed before the OTC is immersed in liquid nitrogen [33,39].

Following cryopreservation, ovarian tissue may be transplanted (in vivo culture), enabling the recovery of both endocrine and gametogenic functions [11]. Alternatively, the tissue can be used in in vitro culture systems to support the development of preantral follicles within the ovarian cortex (Figure 1) [41], or for the culture of isolated follicles retrieved from previously cryopreserved fragments, which may progress to maturity and yield fertilizable oocytes [24]. Moreover, preantral follicles isolated directly from ovarian tissue and subsequently cryopreserved may also progress to the antral stage, producing viable oocytes suitable for in vitro embryo production [42].

3. Ovarian Tissue Transplantation

The ovarian tissue transplantation technique consists of a surgical approach in which an ovarian tissue fragment is transplanted in vivo into a host, where it functions as a biological incubator, stimulating the resumption of folliculogenesis [43]. Unlike in vitro environments, tissue transplantation offers the advantage of providing an optimal environment for follicular activation and development, supplying hormones, growth factors, and biological conditions that in vitro culture systems are not yet fully capable of reproducing [44]. The primary aim of this methodological approach is the restoration of fertility and endocrine activity.

In small ruminants, the first studies on ovarian tissue freezing and transplantation were conducted by Gosden et al. [45] and Baird et al. [46]. Since then, several births in sheep have been reported following the transplantation of frozen hemi-ovaries or whole ovaries [47,48,49], as well as after the transplantation of vitrified ovarian tissue [50,51]. Despite these advances, transplantation as a method for fertility restoration in production species such as sheep and goats remains limited by the need for immunosuppression, the invasive nature of the procedure, and high operational costs, which make its implementation largely unfeasible for small-scale farmers [52]. Table 1 provides a compilation of relevant scientific studies reporting advances in ovarian tissue cryopreservation and transplantation in small ruminants.

Ovarian tissue transplantation can be classified according to several parameters, among which the total size of the grafted material [34], the type of host selected for transplantation [57], and the anatomical implantation site [60] are particularly noteworthy, as summarized in Table 2. In addition to these criteria, factors such as the degree of graft vascularization, the cryopreservation method employed, and the time required for the recovery of ovarian function also play decisive roles in the success of the procedure and are frequently addressed in experimental and clinical studies.

The selection of graft size, host type, and implantation site must be made carefully, as these factors directly influence transplantation success. Graft size affects both the time required for revascularization and the extent of follicular loss due to ischemia: smaller grafts exhibit faster functional recovery, whereas larger grafts, such as whole ovaries, carry a greater ischemic risk if revascularization does not occur promptly [69].

In this context, whole-ovary transplantation emerges as an alternative capable of mitigating some of these effects, as it allows for immediate vascular anastomosis, thereby reducing ischemia–reperfusion injury and promoting preservation of the follicular reserve. However, this approach requires appropriate cryopreservation of the vascular pedicle and involves high surgical complexity [70]. Among the primary mechanisms of follicular loss are acute ischemia, premature activation of primordial follicles, and hypoxia-induced apoptosis. Strategies such as the use of angiogenic factors, antiapoptotic agents, erythropoietin, or co-transplantation of mesenchymal stem cells have been shown to reduce these losses and enhance revascularization [71].

From a clinical standpoint, restoration of endocrine function can be achieved through the transplantation of cortical ovarian tissue fragments or, in specific situations, through whole-ovary transplantation. However, reproductive outcomes vary according to the preservation method, implantation site, and surgical technique employed. Among fragment-based transplants, orthotopic implantation consistently yields the most favorable reproductive results, primarily due to the possibility of permitting natural ovulation and spontaneous pregnancy. Whole-ovary transplantation, although capable of preserving a larger quantity of follicles, remains limited by technical complexity and challenges associated with maintaining the viability of the vascular pedicle [70].

Given these limitations, the use of ovarian cortical fragments has emerged as a promising alternative, as the reduced tissue volume enhances the diffusion of nutrients and oxygen, thereby minimizing ischemia and complications associated with the need for vascular reanastomosis. Additionally, the procedure is less invasive, faster to perform, and allows for the possibility of retransplantation in cases of graft failure [61,72].

4. Maintenance and Follicular Activation: Recent Evidence in Production Species

Studies on ovarian tissue transplantation have highlighted the critical roles of the post-transplant microenvironment, vascular perfusion, and angiogenic factors in follicular survival and activation. Souza et al. [68] evaluated ovaries from mares that were cooled or cryopreserved and subsequently xenotransplanted into mice, demonstrating that cooling—either alone or followed by transplantation—preserved follicular morphology, whereas the cryopreservation–cooling–transplantation sequence markedly reduced follicular survival. Furthermore, transplantation triggered follicular activation, likely as a response to post-surgical hypoxia.

In goats, Vieira et al. [67] examined intra-auricular grafting and observed a higher proportion of healthy and activated primordial follicles seven days after transplantation. However, prolonged ischemia reduced follicular viability, emphasizing the importance of both graft site and post-transplantation interval. In cattle, Morais et al. [73] reported that short-term pre-exposure of ovarian tissue to VEGF before heterotopic autotransplantation enhanced neovascularization but partially affected follicular morphology: transplantation without VEGF resulted in better preservation of primordial follicles, whereas VEGF treatment promoted greater activation of developing follicles. Collectively, these findings indicate that variables such as vascular supply, duration of ischemia, graft location, and angiogenic stimulation interact in a complex and integrated manner to modulate follicular survival, activation, and development following ovarian tissue transplantation.

5. Blood Flow of the Ovarian Tissue Implantation Site

Ultrasonography has proven to be a valuable tool for real-time, non-invasive monitoring of ovarian structures in small ruminants, both in non-stimulated animals [74] and in those subjected to hormonal stimulation [75]. In the context of ovarian tissue transplantation in humans, two-dimensional ultrasonography has been widely used to monitor follicular [63] and embryonic development [76,77].

An important variation of this method, still underexplored in studies involving ovarian transplantation, is color Doppler ultrasonography, which enables real-time visualization of vascular dynamics by detecting the movement of erythrocytes within vessels near the transducer [78]. This approach not only allows identification of anatomical structures but also provides an assessment of blood perfusion, offering essential information regarding post-transplant revascularization, a process that is critical for follicular survival, maintenance, and activation and therefore fundamental to graft success.

In the study conducted by Pinto et al. [79], color Doppler ultrasonography was used to assess local blood flow within and around implanted tissues. The perimeter and number of colored pixels were measured on alternating days over seven days (right side) and fifteen days (left side). The authors observed that blood perfusion was similar among the different regions evaluated around the implants during the first two days following transplantation, suggesting a positive correlation between vascular perfusion and the presence of normal and primordial follicles. These findings reinforce the applicability of color Doppler imaging as a non-invasive tool for monitoring perfusion in superficial grafts.

Complementarily, Vieira et al. [59] performed daily assessments of blood flow from day 0 (pre-transplant) to day 15 (post-transplant) at the graft site in goats subjected to ovarian allotransplantation. The images obtained revealed a progressive and significant increase in vascularization in the analyzed regions, particularly when comparing day 0 with days 7 and 15 after the procedure (Figure 2).

The effectiveness of Doppler ultrasonography as a non-invasive method for evaluating blood flow in ovarian implants has also been demonstrated in other species, including equine [80] and bovine [73]. Taken together, these findings establish color Doppler imaging as an essential tool for monitoring vascularization and the viability of transplanted ovarian tissues, contributing to the advancement of techniques aimed at preserving and restoring reproductive function across different species.

6. Histology and Immunohistochemistry Applied to Ovarian Tissue

The appropriate choice of staining method is essential for accurate histological assessment, as different dyes allow the visualization of specific cellular structures and components of the extracellular matrix [81]. In ovarian tissue, Periodic Acid–Schiff Hematoxylin (PAS) staining is widely used, producing a purple–blue coloration in the stroma and follicular cells and a pink coloration in carbohydrate-rich structures such as the zona pellucida and the basement membrane, thereby facilitating the identification of potential abnormalities [82]. Among these abnormalities are thickening or duplication of the basement membrane, discontinuities or irregularities in the zona pellucida, pyknotic oocytes, granulosa cells exhibiting cytoplasmic degeneration, and stromal alterations such as fibrotic areas or abnormal accumulation of extracellular matrix, which are critical parameters for evaluating tissue integrity and follicular viability after preservation or culture procedures [83].

Another relevant technique is Picrosirius Red staining, which is particularly indicated for the characterization of collagen fibers [84]. Under conventional light microscopy, the total collagen content in the section appears pink, whereas under polarized light, type I collagen fibers exhibit a red coloration and type III fibers appear in shades of green [84]. This approach is useful for identifying regeneration, structural damage, or fibrosis following in vitro culture [85] or transplantation [13], thereby providing complementary information on extracellular matrix remodeling in preserved or grafted ovarian tissue.

Immunohistochemistry (IHC) is based on morphological, molecular, and immunological principles to identify specific antigens in tissues through highly specific antigen–antibody interactions. This technique enables the detection, localization, and semi-quantitative assessment of proteins in cellular and tissue compartments, and it is widely used in both experimental research and diagnostic pathology [86]. In the context of ovarian biology, IHC has been employed to identify proteins expressed in the stroma or parenchyma, allowing the evaluation of key processes such as angiogenesis, apoptosis, and cell proliferation, particularly in tissues subjected to ovarian transplantation [73,87]. A summary of the most used immunohistochemical indicators in studies of transplanted ovarian tissue, highlighting their respective applications is presented in Table 3.

The assessment of blood vessel density is essential for analyzing tissue perfusion and the viability of ovarian grafts. The markers CD31 and α-smooth muscle actin (αSMA) allow the distinction between newly formed and mature vessels, respectively [19,57,73]. Combined analysis of these markers enables the correlation of revascularization with follicular development and stromal integrity, reflecting the efficiency of the graft in supporting adequate diffusion of nutrients and oxygen.

Angiogenic factors such as vascular endothelial growth factor (VEGF) have shown significant potential to improve vascularization in cryopreserved and transplanted ovarian tissue. Experimental studies demonstrate that VEGF supplementation increases the density of newly formed and mature vessels, reduces apoptosis, and contributes to the preservation of primordial follicles after transplantation [73,90,91]. In murine models, the combined use of VEGF and basic fibroblast growth factor (FGF2) further enhanced follicular survival and revascularization, suggesting promising perspectives for clinical application and for use in domestic species of zootechnical interest [19,92].

In addition to angiogenesis, the evaluation of cell proliferation is commonly performed using markers such as Ki-67 [89] and proliferating cell nuclear antigen (PCNA) [73], whereas the follicular reserve can be investigated with germ cell markers such as DDX4 [88]. Increased Ki-67 [89] and PCNA [73] expression in granulosa and stromal cells has been associated with regenerative activity and follicular activation in transplanted tissues treated with VEGF or other modulatory agents. Taken together, these findings reinforce immunohistochemistry as a fundamental approach to evaluate angiogenesis, cellular viability, and the maintenance of ovarian function in preserved and transplanted ovarian tissue, enabling the establishment of robust correlations between therapeutic interventions and graft quality, vascular integration, and follicular survival.

7. Future Perspectives

Emerging investigative approaches driven by recent advances are reshaping the future of ovarian tissue cryopreservation and transplantation, with emphasis on multiple complementary strategies aimed at improving graft performance, follicular preservation, and reproductive outcomes. Current directions highlight the need to: i) accelerate revascularization and reduce post-transplant ischemia by incorporating pro-angiogenic molecules, biomaterials, or cellular therapies; ii) modulate follicular activation to preserve the pool of primordial follicles and avoid premature depletion; iii) optimize cryopreservation protocols through the use of less toxic cryoprotectants and more efficient cooling–warming curves; iv) integrate tissue engineering approaches, including three-dimensional scaffolds and hydrogels capable of supporting the follicular microenvironment; v) apply molecular and omics technologies to precisely map cellular, metabolic, and inflammatory processes associated with thermal and hypoxic injury; vi) explore new implantation sites that are minimally invasive, well vascularized, and capable of sustaining endocrine and reproductive functions; vii) conduct long-term functional evaluations correlating perfusion, morphology, and actual reproductive performance, especially in species of zootechnical and conservation interest; and viii) expand the use of ovarian tissue transplantation in wildlife species, developing species-specific strategies that account for anatomical, physiological, and reproductive differences.

Taken together, these perspectives underscore an expanding field in which the integration of basic and applied science has the potential to enhance the effectiveness of ovarian tissue transplantation in animal production, clinical medicine, and biodiversity conservation. Continued multidisciplinary efforts involving reproductive physiology, biotechnology, tissue engineering, and molecular analysis will be essential for transforming current limitations into viable opportunities for fertility restoration.

8. Conclusion

Ovarian tissue cryopreservation and transplantation have advanced significantly in recent decades, establishing themselves as central strategies for fertility preservation in various species of zootechnical and clinical interest. The success of these techniques depends on meticulous attention across all procedural stages, from tissue collection and processing to transplantation and postoperative evaluation, particularly regarding revascularization, tissue repair, and cellular proliferation.

In this context, Doppler ultrasonography has emerged as a valuable tool for the continuous assessment of blood perfusion in transplanted ovarian tissues, enabling real-time, in vivo monitoring of vascular changes that are essential for graft survival and follicular development. Following tissue recovery, histological analysis combined with immunohistochemical markers remains indispensable for elucidating the underlying biological processes from a structural and molecular perspective. These tools provide detailed insights into tissue remodeling, angiogenesis, apoptosis, and the maintenance of follicular reserves.

Despite significant progress and promising results reported in the literature, current evidence indicates that follicular survival remains limited by complex and still insufficiently understood pathophysiological mechanisms. Continued research is therefore necessary to standardize protocols, refine grafting methodologies, optimize microenvironmental support, and broaden the applicability of ovarian tissue transplantation across domestic and wild species.