Frozen-Thawed ovarian autografts treated with scaffold-based melatonin delivery in rats

Abstract

Introduction

Cryopreserved ovarian tissue transplantation is the only option for fertility preservation in children undergoing cytotoxic treatments. Among antioxidant substances, melatonin has been increasingly investigated to improve the avascular ovarian graft. However, little is known about its effect when applied through an absorbable sponge matrix - a local and non-invasive application approach.

Methods

Thirty adult Wistar rats were divided into three experimental groups (n = 10 each): control (CG), Gelfoam^® (GF), and Gelfoam^® + melatonin (GFM). The animals underwent oophorectomy and both ovaries were subjected to a slow cryopreservation protocol and stored in liquid nitrogen for 24 h. After thawing, the whole ovaries were transplanted into the retroperitoneum without vascular anastomosis and fixed to each side of the psoas muscle with a single non-absorbable suture. Immediately after transplantation, ovarian grafts were treated with Gelfoam^® soaked either in vehicle (GF) or in melatonin at 10⁻⁷ M (GFM). In all groups, the applied vehicle volume was 15 µl. No treatments were administered to the control group. The grafts were recovered after 30 days, and the animals were euthanized with a lethal dose of anesthetic. Histological analyses (follicle and corpus luteum density), fibrosis assessment (collagen fiber types I and II), and immunohistochemistry for endothelial cells (von Willebrand factor), apoptosis (TUNEL), and cell proliferation (Ki67) were performed.

Results

Ovarian follicles at different maturation stages and intact and functional corpora lutea were observed in all groups. Melatonin promoted an increase in endothelial cells (p < 0.05, GFM vs. CG and GF) and reduced cellular proliferation within the ovarian follicles (p < 0.05, CG vs. GFM; Fig. 3) without altering this parameter in the corpora lutea (p > 0.05). Animals in the GF group showed increased cell proliferation in the corpora lutea, which was attenuated by melatonin treatment (GF vs. CG and GFM; p < 0.05). Apoptosis in the corpora lutea was reduced by melatonin treatment (p < 0.05, CG vs. GFM). No differences were observed among the groups in the number of leukocytes, corpora lutea, viable and atretic ovarian follicles, quantification of type I and III collagen fibers or apoptosis in ovarian follicles.

Conclusion

Melatonin delivered via an absorbable sponge improved the viability of cryopreserved ovarian grafts in rats by enhancing endothelial cells and reducing apoptosis and cell proliferation, without inducing an inflammatory response. Future studies should evaluate its effects on human tissues, either alone or in combination with other cytoprotective agents, to optimize ovarian graft survival.

Background

Ovarian tissue transplantation after cryopreservation is the most recent option for fertility preservation, and its use has expanded considerably over the past two decades. Compared with other techniques, it offers several advantages, including not delaying oncologic treatment, can be performed independently of menstrual cycles, is safe for hormone-dependent neoplasms, and restores both fertility and endocrine function. It is also the only fertility preservation method available for children and adolescents undergoing cytotoxic therapy, as it does not require ovarian stimulation or sperm donation. Endocrine recovery occurs in approximately 90% of cases within six months, and pregnancy rates—spontaneous or assisted—reach about 30%. Nearly 290 pregnancies and 160 live births following ovarian tissue transplantation have been reported worldwide [1]. Since 2019, the American Society for Reproductive Medicine no longer considers ovarian tissue cryopreservation experimental, enabling its use in prepubertal patients or in situations where time is insufficient for ovarian stimulation [2]. In such cases, established fertility preservation methods such as embryo or oocyte cryopreservation are not feasible [3, 4].

With safety and efficacy established, the main challenge is to prolong graft longevity, which directly depends on follicular density [5]. Experimental work indicates that up to two-thirds of cortical oocytes are lost during the revascularization phase after avascular transplantation [6–9]. To accelerate this process—typically beginning 4–5 days after grafting—multiple strategies have been explored [10], including refined surgical techniques [11–13], pro-angiogenic and antioxidant agents [14–18], and stem cell–based approaches [19–21].

Among antioxidant agents, melatonin has received particular attention in experimental ovarian transplantation and fertility preservation [22]. Its protective effects on follicles at different maturation stages are well documented [21, 23]. High intrafollicular melatonin concentrations help prevent atresia and reduce oxidative stress, and levels in follicular fluid correlate with oocyte quality, supporting its use in oocyte selection [24, 25]. Melatonin also attenuates age-related fertility decline by reducing mitochondrial oxidative stress [26]. In transplanted ovaries, it limits oxidative injury, necrosis, and apoptosis when administered orally or intraperitoneally [16, 27, 28]. Our group previously showed that adding melatonin to the cryoprotectant medium increases mature follicle counts, enhances type I collagen deposition, angiogenesis, and proliferation, and reduces apoptosis [29]. More recent findings demonstrate that cryopreservation with melatonin leads to improved hormonal profiles (including lower follicle-stimulating hormone [FSH] levels) and higher expression of antioxidant enzymes such as superoxide dismutase 2 (SOD2), suggesting reduced tissue damage during cryopreservation and transplantation [30]. However, it remains unknown whether the benefits observed during cryopreservation can be reproduced when melatonin is applied directly to the avascular graft.

Gelfoam^® is an acellular porcine-derived gelatin matrix characterized by softness, high porosity, adaptability to tissue surfaces, and slow absorption, typically over four weeks. It is widely used as a hemostatic agent and is considered safe for clinical use [31]. In experimental ovarian transplantation, it provides a safer alternative to direct parenchymal injection [32], serving as a scaffold for non-invasive graft treatment. This matrix enables gradual and continuous release of therapeutic agents without causing morphofunctional damage and is fully absorbed within four weeks [20]. Incorporating an antioxidant such as melatonin into a Gelfoam^® scaffold may therefore reduce oxidative damage and enhance vascularization in transplanted ovarian tissue. Thus, this study aimed to evaluate the effects of melatonin applied directly to ovarian grafts using an absorbable sponge matrix.

Methods

This study is part of an ongoing research program in fertility preservation and was approved by the Ethics Committee on Animal Use of the Faculdade de Medicina, Universidade de São Paulo (FMUSP) on March 25, 2015 (protocol 024/15). All procedures were conducted at the Laboratory of Medical Investigation, Department of Obstetrics and Gynecology, FMUSP, in collaboration with the Laboratory of Genetics and Molecular Cardiology.

Thirty intact female Wistar rats (Rattus norvegicus albinus), weighing 200–250 g, were used. The animals received species-appropriate chow and potable water ad libitum throughout the experiment and were maintained under controlled sanitation, lighting, and temperature conditions at the FMUSP animal facility.

Experimental design

Thirty intact female Wistar rats (200–250 g) were maintained under controlled conditions with free access to chow and water. Animals were randomly assigned to three groups (n = 10 each): control (CG), Gelfoam^® (GF), and melatonin-treated Gelfoam^® (GFM). In GF and GFM, a Gelfoam^® sponge (≈ 2 mm²) was placed over each ovarian graft, soaked with either vehicle (Phosphate-Buffered Saline - PBS) or melatonin (10⁻⁷ M; 15 µL). No treatment was applied in CG (Fig. 1).

Fig. 1.

Study experimental design

Vaginal cytology

Daily vaginal smears were collected between 8:00–10:00 a.m. during the preoperative phase. Only rats with at least two regular 4-day cycles were included. Smears were obtained with saline-moistened swabs, fixed in ethanol, stained by Shorr–Harris, and examined at 10×/40× magnification [33]. Transplantation occurred during diestrus. From postoperative day (POD) 4 to euthanasia (POD 30–35), smears were again collected daily during diestrus [18–20]. Ovarian transplantation was performed during the diestrus phase.

Bilateral oophorectomy

Once the diestrus phase was confirmed, the animals were anesthetized using intraperitoneal injections of xylazine and ketamine at doses of 15 and 60 mg/kg, respectively. The abdominopelvic cavity was opened to identify the ovaries. The ovarian pedicles were clamped and immediately released using 4 − 0 nylon sutures. The uterine tubes were resected along with the periovarian adipose tissue, and the ovaries were immediately processed for slow cryopreservation. The abdominal wall was closed in two layers (musculoaponeurotic/peritoneal and skin) using 5 − 0 mononylon sutures.

Ovarian tissue cryopreservation

After bilateral oophorectomy, fresh intact ovaries were cryopreserved using a slow-freezing freezer (CL-8800, Cryogenesis Software, Freezer Control), as previously described [34]. Ovaries were placed in 1.8mL cryovials (Sigma-Aldrich^®, Inc.) containing M2 medium with HEPES (Sigma-Aldrich^®, Inc.) and 1.4 M dimethyl sulfoxide as cryoprotectant, and maintained at room temperature for 5 min.

Cryovials were sealed and placed in the programmable freezer, cooled from 25 °C to 10 °C at a rate of 1 °C/min, then to − 7 °C at 0.5 °C/min, and held at − 7 °C for 5 min. Ice nucleation was manually induced using precooled forceps, and temperature was maintained at − 7 °C for another 5 min to release latent fusion heat. The samples were then cooled to − 55 °C at 0.5 °C/min, plunged into liquid nitrogen (–196 °C), and stored for 24 h.

For thawing, the cryovials were removed from liquid nitrogen and kept at room temperature until the ice melted. The ovaries were washed twice for 5 min in fresh M2 medium, gently agitated to remove the cryoprotectants, and maintained in M2 medium at room temperature until transplantation.

Autologous transplantation of cryopreserved ovaries

Twenty-four hours after cryopreservation, the animals underwent a second laparotomy for ovarian transplantation. Each animal received its own pair of ovaries (autologous transplantation). Immediately after thawing, both intact ovaries were fixed to the retroperitoneum (heterotopic transplantation), one on each side of the psoas muscle near the aorta and vena cava, using a single 4 − 0 mononylon stitch without vascular anastomosis.

Melatonin application to ovarian grafts

The melatonin was used in a 10⁻⁷M concentration (Sigma Aldrich, Saint Louis, MO, USA), as standardized in previous studies [29, 35]. In the GFM group, the 2 mm² Gelfoam^® sponge was soaked with melatonin and immediately applied to the surface of both ovarian grafts fixed in the retroperitoneum. In the GF group, the same procedure was performed using Gelfoam^® soaked in PBS, the same vehicle used for melatonin preparation. The abdominal wall was closed in two layers with 5 − 0 mononylon sutures, including the musculoaponeurotic/peritoneal layer and the skin.

Graft retrieval and histological preparation

On predetermined euthanasia days (between Postoperative Day 30 and 35 and after identification of the estrus phase), the animals were anesthetized as described previously for sample collection and euthanasia. All the animals were euthanized on the first time which estrus phase was identified after POD 30. An incision was made over the previous scar to locate the ovarian grafts, which were subsequently removed whole. After sample collection, euthanasia was performed using a lethal dose of the previously used anesthetics.

Both ovarian grafts were immediately fixed in 4% paraformaldehyde for at least 24 h. Following fixation, the ovaries were dehydrated, paraffin-embedded, serially sectioned at 5 μm, and mounted on glass microscope slides. Routine hematoxylin and eosin (HE) staining was performed for histological examination with light microscopy. The ovarian cortex was sectioned into two pieces of equal size, and five representative sections were selected.

The following analyses were performed: (1) morphological: macroscopic and histological; (2) morphometric: classification and counting of ovarian follicles (immature and mature), corpora lutea, and blood vessels; and (3) immunohistochemical: assessment of neoangiogenesis (von Willebrand factor - VWF), apoptosis (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling -TUNEL), and cell proliferation (Ki-67).

Morphological and morphometric analyses

To evaluate follicular development, ovarian follicles were counted and classified into two groups: developing (at any maturation stage) and atretic follicles. Developing follicles were further classified as immature (primordial, primary, and secondary) or mature (single large antral follicle), in addition to the corpora lutea. Both viable and atretic follicles as well as functional and degenerating corpora lutea were considered [36].

Atretic follicles were defined by wrinkled zona pellucida, disorganized granulosa cells, and pyknotic nuclei, regardless of their size. Viable follicles showed no degenerative changes and were classified as follows: primordial (oocytes surrounded by a single layer of flattened cells), primary (oocytes surrounded by one or more layers of cuboidal or columnar cells without an antrum), secondary (presence of oocytes and antrum), and mature (oocytes with a large antrum) [35].

Functional corpora lutea were characterized by intact luteal cells with large nuclei and surrounding capillaries, whereas degenerating corpora lutea displayed leukocyte and macrophage infiltration, particularly neutrophils. Inflammatory infiltrates were considered intense when leukocytes outnumbered the normal tissue cells [36].

All images of the sections were obtained using an image acquisition software system (Leica DM2500; LEICA, Wetzlar, Germany), and measurements were made using Leica QWin V3 software. Counts were performed in four fields per animal at 10x magnification by two independent investigators blind to the experimental treatments performed all of the analyses under a microscope (LEICA).

Immunohistochemistry assays

Sections containing ovarian tissue were immunostained to measure neoangiogenesis using von Willebrand factor for endothelial cells (AB6994, 1:100,

Abcam Inc., Cambridge, MA, USA), cell proliferation using Ki-67 expression (M724001-2, 1:100, Dako North America Inc., Carpinteria, CA, USA) and apoptosis using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay.

Cross sections embedded in paraffin were treated with antigenic exposure and blocked with 2% casein in PBS. The tissue sections were incubated with the respective primary antibodies overnight at 4 °C. The sections were incubated with a biotinylated rabbit secondary antibody (universal Polymer anti-mouse and rabbit Histofine^® 1:400, Vector Laboratories, Burlingame, CA, USA) and streptavidin peroxidase followed by the peroxidase substrate diaminobenzidine tetrahydrochloride according to the manufacturer’s instructions. The tissue slices for the TUNEL assay were stained using a commercially available kit (In Situ Cell Death Detection Kit, Fluorescein, Roche, Germany, 11684795910) following the manufacturer’s instructions. Red-brown coloring of the cytoplasm/nucleus was considered positive staining (any other coloring was considered negative staining). Negative controls were used to avoid bias. For the negative controls, the primary antibody was omitted in each different immunohistochemistry staining.

Images of the sections were obtained using an image acquisition software system (Leica DM2500), and measurements were made using Leica QWin V3 software. A red-brown coloring of the cytoplasm/nucleus cell expression was specified as positive staining (otherwise as negative staining). A positive cell staining assessment was performed in eight different fields per animal at 200x magnification, and the results were expressed as the percentage of positive area (arbitrary units/mm²) [19, 20, 32]. All measurements were conducted by two independent investigators who were blinded to the experimental groups.

Statistical analysis

According to the Shapiro-Wilk normality test, One-way ANOVA (for normal distribution) or Kruskal–Wallis tests (for non-normal distribution) were utilized to compare the treatment groups (Control, GF and GFM). One-way ANOVA followed by Tukey’s post-hoc test or Kruskal–Wallis ANOVA followed by Dunn post-hoc test was applied.

The results were expressed as the mean ± standard deviation of the mean (one-way ANOVA) or median (Kruskal–Wallis). All statistical analyses were performed using GraphPad Prism 10.6.1 (892) (GraphPad Software Inc, San Diego, CA, USA). P values lower than 0.05 (5%) were considered significant.

Results

In vaginal cytology, all animals from both experimental groups exhibited regular estrous cycles in the estrus phase, indicating ovulation, and consequently, the functional activity of the ovarian grafts.

Morphological analysis revealed that the ovarian grafts in both groups had preserved morphology, with ovarian follicles at various stages of development. In both groups, ovarian follicles at different maturation stages, and intact and functional corpora lutea were observed. The number of leukocytes in the graft tissue did not change with the treatment (Fig. 2).

Fig. 2.

Photomicrographs of rats cryopreserved ovarian grafts. Hematoxylin–Eosin staining. A Control, B GF, and C GFM. Magnification 50x. CL, corpus luteum; F, follicles; TG, granulation tissue; M, muscle; arrows, blood vessels. D Mean leukocyte Count. ns, non-significant, One-Way ANOVA

Melatonin promoted an increase in endothelial cells (p < 0.05, GFM vs. CG and GF; Fig. 3) and reduced cellular proliferation within the ovarian follicles (p < 0.05, CG vs. GFM; Fig. 4) without altering this parameter in the corpora lutea (p > 0.05; Fig. 3). Animals in the GF group showed increased cell proliferation in the corpora lutea, which was attenuated by melatonin treatment (p < 0.05, GF vs. CG and GFM; Fig. 4). Apoptosis in the corpora lutea was reduced by melatonin treatment (p < 0.05, CG vs. GFM; Fig. 5). All these results are shown in Table 1.

Fig. 3.

Photomicrographs of cryopreserved ovarian grafts from rats: immunohistochemistry for endothelial cells (von Willebrand factor – factor VIII) in the stroma. A CG, B GF, and C GFM. Dark brown-stained cells are considered positive. Magnification 200x. D Quantification of positive cells; results are expressed as the percentage of positive cell area (arbitrary unit/mm²). ns, non-significant; *p < 0.05; One-Way ANOVA on ranks complemented by Tukey test

Fig. 4.

Photomicrographs of cryopreserved ovarian grafts from rats: immunohistochemistry for cell proliferation (Ki-67) in ovarian follicles. A Control, B GF, and C GFM. Dark brown-stained cells are considered positive. Magnification 200×. D Quantification of positive cells in ovarian follicles and E Results are expressed as the percentage of positive cell area (arbitrary unit/mm²). ns, non-significant; *p<0.05; **p<0.01; ***p<0.001. One-Way ANOVA on ranks complemented by Tukey test

Fig. 5.

Photomicrographs of cryopreserved ovarian grafts from rats: immunohistochemistry for apoptosis (TUNEL) in corpora lutea. A Control, B GF, and C GFM. Dark brown-stained cells are considered positive. Magnification 200×. D Quantification of positive cells; results are expressed as the percentage of positive cell area (arbitrary unit/mm²). ns, non-significant; *p<0.05; One-Way ANOVA on ranks complemented by Tukey test

Table 1.

Immunohistochemical analyzes of rat frozen-thawed ovarian grafts treated or not with melatonina delivered by an absorbable sponge

	Control	GF	GFM
vWF	1.13 ± 0.58	1.08 ± 0.51	1.92 ± 0.78*
Ki-67 Fol	1.46 ± 0.70	0.94 ± 0.67	0.40 ± 0.49
Ki-67 CL	1.671 ± 0.93	3.118 ± 0.71	0.99 ± 0.55
TUNEL	0.395 ± 0.37	0.121 ± 0.19	0.135 ± 0.28

vWF (von Willebrand factor): p<0.05 GFM vs. Control and GF. Ki-67 Fol (follicle): p<0.05, Control vs. GFM. Ki-67 CL (Corpora lutea): p<0.05, GF vs. CG and GFM. TUNEL: p<0.05, CG vs. GFM. Data were shown in mean ± SD. One-way ANOVA test

No differences were observed among the groups in the quantification of type I and III collagen fibers, number of corpora lutea, viable and atretic ovarian follicles, or apoptosis in ovarian follicles (Table 2).

Table 2.

Morphometric analyses of rat frozen-thawed ovarian grafts treated or not with melatonin delivered by an absorbable sponge

	Control	GF	GFM
Leukocytes	37.1 ± 28.7	39.2 ± 34.8	43 ± 25.9
Collagen type I	26.1 ± 15.5	27.7 ± 14.1	22.2 ± 6.13
Collagen type III	22.1 ± 9.3	23.9 ± 7.5	15.3 ± 9.05
Immature follicles	4 ± 1.51	3 ± 2.16	4.66 ± 2.04
Mature follicles	3 ± 1.82	2.25 ± 1.5	1.6 ± 0,54
Atretic follicles	1.4 ± 1.34	0.8 ± 1.3	0.6 ± 1.34
Intact corpora lutea	7 ± 4.84	6.33 ± 4.27	5.57 ± 5.34

p>0.05, one-way ANOVA

Discussion

Ovarian tissue transplantation is a currently available technique for preserving fertility in patients undergoing oncological treatments or those with other conditions that lead to premature ovarian insufficiency. Among the established methods, such as follicle and embryo cryopreservation, this is the most recent, with growing interest over the past two decades. It is no longer considered experimental by some reproductive societies [37]. The wide heterogeneity of protocols described in the international literature, both for cryopreservation and graft implantation, remains a barrier to overcome and limits the reproducibility of findings.

Oxidative damage during cryopreservation is of particular concern, because freezing induces cellular oxidative stress, generating reactive oxygen species that can cause irreversible damage or apoptosis [38]. This study aimed to explore the antioxidant and anti-apoptotic properties of melatonin to mitigate the negative effects of cryopreservation and reimplantation of ovarian tissue. Rodent experimental models are particularly suitable for evaluating fertility and reproduction because of their short estrous cycle, averaging 4–5 days [36].

The fluid bathing human follicular cells contains significantly elevated concentrations of melatonin [39], derived not only from ovarian [40] but also from systemic production [41]. This presence and activity of melatonin protects oocytes from oxidative stress within the follicular fluid [24] and can directly influence oocyte quality. Melatonin supplementation in women with diminished ovarian reserves can increase reduced glutathione levels and total antioxidant capacity, indicating decreased follicular oxidative stress [42].

The present study demonstrated that melatonin delivered through a sponge-based absorbable matrix (Gelfoam^®) could increase the viability of cryopreserved ovarian grafts, promoting endothelial cell proliferation and reducing apoptosis in corpora lutea. These findings align with previous research showing the antioxidant and angiogenic properties of melatonin [22, 24, 25, 30] but uniquely highlight its efficacy when applied locally to grafts in a non-invasive manner, bypassing systemic metabolism.

The mild reduction in leukocytes in ovaries treated with only Gelfoam may be attributed to two main mechanisms: (a) the absorbable sponge, Gelfoam^®, may have acted as a physical barrier even without melatonin (GF group), limiting inflammatory cell infiltration into the ovarian graft. Previous studies have shown that porous matrices such as Gelfoam^® reduce initial inflammatory responses by minimizing direct contact between transplanted tissue and the hostile receptor environment [18]. This property is especially relevant in avascular transplants, where ischemia-reperfusion triggers an acute inflammation cascade [7]; (b) the anti-inflammatory action of melatonin may have further reduced leukocyte infiltration through its known immunomodulatory and antioxidant properties [22, 24], as melatonin suppresses proinflammatory cytokine release (e.g., IL-6, TNF-α) [28] and inhibits neutrophil migration to injured tissue, thereby attenuating post-ischemic oxidative stress [25]. Additionally, this indoleamine reduces the expression of adhesion molecules (e.g., ICAM-1), limiting leukocyte recruitment [11].

The significant increase in endothelial cells (marked by the Von Willebrand factor, Fig. 3) in the melatonin-treated grafts suggests enhanced neovascularization of the cryopreserved ovarian tissue. The von Willebrand factor is a key marker of endothelial activation and vessel formation, and its higher expression in the GFM group indicates that melatonin potentiated post-transplant angiogenesis [15].

Previous studies have demonstrated that melatonin stimulates vascular endothelial growth factor release and reduces oxidative stress, creating a microenvironment conducive to endothelial migration and proliferation [43]. Moreover, melatonin modulates hypoxia-inducible factor 1α, which is essential for graft adaptation to temporary ischemia [8].

Studies using melatonin in cardiovascular tissues have also shown that melatonin promotes angiogenesis by activating the AKT signaling pathway, suppressing caspase formation, inhibiting reactive oxygen species generation, and inducing antioxidant enzymes such as catalase [44]. Liu et al. (2020) [45] demonstrated that melatonin-treated mesenchymal cells modulated inflammation and accelerated wound healing through increased angiogenesis and collagen synthesis in diabetic rats. Therefore, the elevated Von Willebrand factor expression in the GFM group not only reflects improved revascularization but also suggests accelerated functional recovery of the transplanted tissue, optimizing follicular survival and hormonal function [4]. These findings support the potential clinical use of melatonin in biodegradable matrices to improve ovarian tissue transplantation outcomes, particularly in patients at risk of post-graft vascular failure [46].

The significant reduction in apoptosis observed in the GF group (Gelfoam^® + vehicle, Fig. 5) compared with the Control Group may be explained by the physical and biochemical properties of the scaffold itself. Gelfoam^®, an absorbable gelatin sponge, acts as a three-dimensional matrix that: (1) protects against mechanical stress by minimizing cellular injury during transplantation, thus reducing the release of pro-apoptotic factors (e.g., caspases) associated with tissue handling [19]; (2) modulates the microenvironment by retaining endogenous trophic factors (e.g., IGF-1) that inhibit apoptotic pathways [29]; (3) serves as a physical barrier against inflammatory infiltration, as the concurrent reduction in leukocytes in the GF group (Fig. 1) suggests decreased activation of pro-apoptotic cytokines (e.g., TNF-α), as shown in similar transplantation models [11].

Although the GFM group (Gelfoam^® + melatonin) exhibited a more pronounced effect, the difference between GF and Control Group highlights that the biomaterial scaffold itself contributes to cellular protection independent of melatonin. These findings underscore the importance of matrix selection (in this study, an absorbable sponge) in experimental models designed to test new tissue preservation strategies.

An intriguing and tissue-specific effect was observed regarding cell proliferation within the corpora lutea. While melatonin reduced proliferation in the follicular compartment—a potentially beneficial effect indicating reduced stress-induced hyperproliferation—the Gelfoam^® scaffold alone (GF group) significantly increased Ki-67 + cells in the corpora lutea compared to both the control and the melatonin-treated groups. This suggests that the acellular matrix itself may create a provisional microenvironment that stimulates luteal cell activity, possibly by serving as a reservoir for endogenous mitogenic factors or by modulating local inflammatory signals that are known to influence luteal function (Citing relevant luteal biology/transplantation refs if available). Importantly, this scaffold-induced proliferative response was attenuated by melatonin in the GFM group. Melatonin is known to exert potent anti-proliferative and pro-differentiation effects in various steroidogenic tissues through receptor-mediated pathways and antioxidant mechanisms [22, 24]. In the context of transplantation, this modulating effect of melatonin on scaffold-driven proliferation may reflect a normalization of luteal dynamics, preventing potential hyperplasia or aberrant tissue remodeling. Although the functional consequence of this moderated proliferation on long-term CL steroidogenesis requires further study, it highlights the complex and compartmentalized responses within the ovarian graft to combined biomaterial and pharmacological interventions.

This study has several strengths. This is the first study to test melatonin delivery via a matrix-based (absorbable sponge) system in ovarian grafts, offering a novel targeted therapeutic strategy. It employed rigorously standardized methods developed over a decade by our group, including controlled cryopreservation, blinded histomorphometry, and a standardized melatonin concentration (10⁻⁷ M) [12, 18, 19, 29]. This study also demonstrates translational relevance, as it mimics clinical scenarios of avascular transplantation [37].

However, its limitations must also be considered: it only employed a short-term follow-up (30 days), as long-term graft function and fertility outcomes were not evaluated; we used an animal model, given the differences in follicular dynamics and hypoxia tolerance compared with humans [9]; and mechanistic data—potential molecular pathways (e.g., MT1/MT2 receptor activation) were not explored. Furthermore, our experimental design included a control for the scaffold effect (GF group) but did not include a group receiving melatonin solution applied directly to the graft without the Gelfoam^® matrix. This omission limits our ability to distinguish whether the benefits observed in the GFM group are due to melatonin’s pharmacological action per se, the sustained local release and protective microenvironment provided by the scaffold, or a combination of both. While the superior outcomes in GFM compared to GF suggest a specific melatonin effect, we cannot conclusively determine if the scaffold-based delivery offers advantages over a simpler topical application. This represents an important direction for future research, as comparing these delivery methods will be crucial for clinical translation in ovarian tissue transplantation.

The melatonin delivery system based on an absorbable sponge may advance treatment strategies aimed at improving ovarian tissue graft survival, particularly in pediatric cancer patients exposed to gonadotoxic therapies [46]. By enhancing graft vascularization, this approach may improve hormonal function and follicular survival, reducing the need for repeated transplantations [47]. Furthermore, local melatonin administration may minimize the systemic side effects (e.g., drowsiness) commonly associated with oral use [21].

Other cytoprotective agents, such as vascular endothelial growth factor, have been used in cryopreservation media for ovarian tissue and may be considered for future studies, either in comparison or in combination with melatonin [15]. Another example is metformin, which has demonstrated anti-apoptotic and pro-angiogenic properties, as well as improved cryopreservation efficiency [48]. Future trials should explore optimal dosing and combination therapies with such agents to further enhance ovarian graft longevity. Although additional research is needed to validate these findings in human tissues and long-term models, this study establishes a foundation for a low-risk, non-invasive, and potentially impactful adjuvant therapy for female fertility preservation.

Conclusion

Melatonin delivered via an absorbable sponge improved the viability of cryopreserved ovarian grafts in rats by enhancing endothelial cells and reducing apoptosis and cell proliferation, without inducing an inflammatory response. Future studies should evaluate its effects on human tissues, either alone or in combination with other cytoprotective agents, to optimize ovarian graft survival.

Abbreviations

FSH

Follicle-stimulating hormone

SOD2

Superoxide dismutase 2

FMUSP

Faculdade de Medicina, Universidade de São Paulo

POD

Postoperative day

CG

Control group

GF

Gelfoam^® group

GFM

Melatonin Gelfoam^® group

PBS

Phosphate-Buffered Saline

VWF

von Willebrand factor

TUNEL

Terminal deoxynucleotidyl transferase dUTP Nick End Labeling

Authors’ contributions

LDD: Conceptualization, Data curation, Formal analysis, Methodology, Investigation and Writing manuscript. MAS: Data curation, Formal analysis and Writing manuscript. LGJ, ACHS and AETSC: Methodology. NMB and MES: Datacuration, Investigation and Methodology; ECB and JEK: Project administration, Supervision and review/editing manuscript. PC: Validation and review/editing manuscript. JMS: Conceptualization, Funding acquisition, Writing original manuscript and Validation. All the authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), 2018/24224-9 and Scientific Initiation Scholarship supported by Programa Institucional de Bolsas de Iniciação Científica (PIBIC) 2024/2025, Universidade de São Paulo (USP) e Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

This study is part of an ongoing research program in fertility preservation and was approved by the Ethics Committee on Animal Use of the Faculdade de Medicina, Universidade de São Paulo (FMUSP) on March 25, 2015 (protocol 024/15).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.