Introduction
In pediatric cancer cases, including adolescents and young adults (AYA), chemotherapy and radiotherapy can impair ovarian function, leading to infertility. Therefore, fertility preservation may be necessary. Three fertility preservation options are available before treatment: oocyte cryopreservation, embryo cryopreservation, and ovarian tissue cryopreservation. However, only ovarian tissue cryopreservation can be performed immediately and without ovarian stimulation. This is the only option for prepubertal girls and is indicated in AYA women when delaying cancer treatment for ovarian stimulation is infeasible or when insufficient oocytes or high-quality embryos are obtained. The procedure involves the laparoscopic removal of one ovary, cryopreservation of the tissue, and later transplantation of thawed tissue to restore fertility naturally or via in vitro fertilization [1-4].
Two established cryopreservation techniques—slow freezing and vitrification—are used clinically. Historically, slow freezing was implemented first, followed by vitrification. Vitrification, sometimes called ‘ultra-rapid freezing,’ employs high concentrations of cryoprotectants to achieve immediate freezing [5]. The high cryoprotectant concentration used in vitrification lowers the freezing point, reducing tissue damage caused by ice crystal formation [6]. Furthermore, because vitrification does not require a programmable freezer, it is highly cost-effective. For these reasons, vitrification is increasingly being adopted in clinical settings as an alternative to slow freezing.
Additionally, two types of preservation containers exist for frozen ovarian tissue: open and closed systems [7,8]. In closed-system vitrification, ovarian tissue is placed on a highly thermally conductive metal plate, sealed in a pouch, and immersed in liquid nitrogen [7]. In the open method, cryopreserved specimens in a liquid nitrogen tank remain directly exposed, increasing the risk of cross-contamination by viruses, bacteria, other pathogens, and external contaminants. The closed method significantly reduces these risks by preventing direct exposure to liquid nitrogen. Guidelines recommend closed-system vitrification to prevent cross-contamination in liquid nitrogen [9].
Although clinical evidence regarding ovarian tissue cryopreservation is increasing, considerable follicle loss during freezing, thawing, and transfer remains the greatest technical barrier [10,11]. Previous basic research on ovarian tissue cryopreservation has shown that rather than direct apoptosis of primordial follicles, stimulation from ovarian tissue freezing causes excessive development of primordial follicles into primary and secondary follicles, significantly reducing the primordial follicle pool [12]. This phenomenon is known as ‘burnout’ [13].
Mechanistic target of rapamycin (mTOR) is a conserved serine/threonine kinase that integrates nutrient, growth factor, and energy signals to coordinate protein synthesis, cell growth, metabolism, and overall cellular homeostasis [14]. Among these functions, mTOR-mediated control of cell growth is directly involved in folliculogenesis, and the pathway is considered the primary regulator of primordial follicle activation. Excessive mTOR activation leads to rapid depletion of the primordial follicle pool [15,16]. Reports indicate that mTOR inhibitors display promise in protecting ovarian reserve against follicle loss induced by chemotherapeutic agents [17,18]. Additionally, studies have indicated that the primary cause of follicle loss in ovarian tissue cryopreservation is mTOR pathway activation, and follicle protection using mTOR inhibitors is effective in both slow freezing and open-system vitrification methods [12,13,19-22]. However, as noted previously, research on ovarian protectants in closed-system vitrification, which is increasingly used in clinical practice, is lacking. Importantly, meta-analyses of clinical studies have reported that tissue damage differs according to the cryopreservation method used [23,24], and even with the same ovarian protectant, data extrapolation across different cryopreservation methods remains challenging.
This study aims to evaluate whether the mTOR pathway is involved in follicular loss during ovarian tissue vitrification, a method rapidly becoming widespread in clinical practice, and whether the mTOR inhibitor rapamycin can prevent the decrease in primordial follicles. Additionally, it seeks to clarify whether previous basic research findings can be applied to current clinical approaches.
Methods
1. Animal experiments and ethics
All animal procedures were performed in strict compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and the Act on Welfare and Management of Animals in Japan. The procedures were approved by our institution’s Institutional Animal Care and Use Committee (approval No. 2024-5-9). Institute of Cancer Research (ICR) mice were purchased (SLC Inc.) and housed under standard conditions with a 12-hour light/dark cycle. The mice were provided a standard pellet diet and allowed free access to water. After 1 week of acclimatization, 4-week-old mice were euthanized by cervical dislocation under deep anesthesia induced by 5% isoflurane. Subsequently, the ovaries were excised and processed as described below.
2. Rationale for rapamycin concentration
When administered orally for immunosuppressive or antitumor purposes in clinical practice, rapamycin achieves effective serum concentrations of 5 to 20 ng/mL [25-27], corresponding to approximately 20 nM. However, in vivo, tissue concentrations of rapamycin can be more than 40 times higher than serum concentrations [28,29]. Thus, tissue concentrations may potentially reach 800 nM or higher. In this study, we selected a conservative concentration of 750 nM, below the theoretical maximum tissue concentration anticipated in clinical settings.
3. Procedure for evaluating the effects of cryopreservation and thawing on ovaries
To investigate the effects of cryopreservation and thawing, one ovary was taken from each of four mice to serve as the fresh (non-cryopreserved) control group. Another four mice had both ovaries harvested and randomly allocated into two groups: a rapamycin-free non-culture group and a rapamycin-treated non-culture group. In the rapamycin-treated group, 750 nM rapamycin was added to the cryopreservation solution. After thawing, samples in these two groups were immediately processed without further culture. Subsequent analyses included mTOR pathway, apoptosis, and follicular density assays (Figure 1). All cryoprotectant and warming solutions were prepared in 0.0075% (v/v) dimethyl sulfoxide (DMSO), with or without rapamycin.
4. Procedure for evaluating the effects of cryopreservation, thawing, and culture on ovaries
To explore long-term effects, both ovaries from five mice were harvested and randomly allocated into two groups: a rapamycin-free culture group and a rapamycin-treated culture group. The ovaries were cryopreserved, thawed, and cultured for 5 days. mTOR pathway, follicle count, and follicle development assays were then performed (Figure 1).
5. Ovarian tissue cryopreservation, thawing, and culture
After three rinses in sterile phosphate-buffered saline, the ovaries were temporarily incubated in minimum essential medium alpha (MEMα; Gibco Thermo Fisher Scientific) supplemented with 1% penicillin-streptomycin (Gibco Thermo Fisher Scientific), with or without 750 nM rapamycin (LC Laboratories). A closed vitrification kit (VT301; Kitazato Corporation) was used according to a protocol equivalent to human ovarian tissue cryopreservation. The equilibration phase involved two steps (25 minutes and 15 minutes) in two solutions provided in the VT301 kit, in cryoprotectant solution with or without 750 nM rapamycin, prior to closed-system vitrification. A film was placed over the ovarian sections to ensure complete coverage, followed by sealing to exclude air. The device was placed in a pouch, which was gently pressed to expel air and sealed with a heat sealer along the indicated line. Using tweezers, the device was then immediately immersed in liquid nitrogen, ensuring rapid cooling. The ovarian tissues were cryopreserved for at least 2 days before thawing.
A thawing kit (VT302; Kitazato Corporation) was used according to a protocol equivalent to human ovarian tissue thawing. The device was removed from the storage tank and submerged in a liquid nitrogen container. While still immersed, the pouch was cut open, and the device was quickly transferred to an initial thawing medium at 37 °C with or without 750 nM rapamycin for 1 minute, ensuring only ovarian tissue sections were immersed to maintain the medium temperature. Once detached from the device, the tissue underwent three additional thawing steps (5, 10, and 10 minutes) in solutions from the VT302 kit, with or without 750 nM rapamycin.
Thawed ovarian tissues were cultured in MEMα supplemented with 1% penicillin-streptomycin, with or without 750 nM rapamycin, for 5 days. Half of the medium was renewed every other day.
Rapamycin was dissolved in 3 µL DMSO and diluted into 40 mL medium, yielding final concentrations of 0.0075% (v/v) DMSO (3/40,000) and 750 nM rapamycin. To match solvent conditions, the rapamycin-free control medium received the same volume (3 µL) of DMSO alone.
6. Ovarian tissue processing
Ovaries were fixed in 4% paraformaldehyde for 24 hours, dehydrated, and paraffin-embedded with the long axis oriented horizontally to maximize cross-sectional area. For total follicle count analysis, each ovary was serially sectioned at a thickness of 5 µm with 25-µm intervals. Mid-ovary sections (5 µm) were selected for immunostaining, including mTOR pathway, apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]), and follicle density analyses. Slides were digitized using a virtual slide scanner (NanoZoomer; Hamamatsu Photonics), and the images were used for follicle counting and ovarian cross-sectional area calculation.
7. mTOR pathway assay (pS6K immunostaining)
Phosphorylated S6K (pS6K) is a marker for downstream activation of the mTOR pathway. Paraffin-embedded sections were deparaffinized, rehydrated in xylene and ethanol, and heated in ethylenediaminetetraacetic acid (EDTA) buffer (pH 9.0) in a microwave for 10 minutes. Endogenous peroxidase activity was blocked using 3% H2O2 for 10 minutes at room temperature. After nonspecific binding was blocked for 20 minutes, the sections were incubated overnight at 4 °C with anti-phospho-rpS6 antibody (#2211; Cell Signaling Technology) at a 1:800 dilution. Secondary antibody incubation was performed for 1 hour at room temperature, followed by diaminobenzidine (DAB) staining. A paraffin-embedded kidney section served as the positive control.
To quantify the percentage of primordial follicles positive for pS6K, a follicle was considered positive if its staining intensity exceeded that of primordial follicles from the fresh-control group. The percentage was calculated as the number of pS6K-positive primordial follicles divided by the total number of primordial follicles in each ovarian section.
8. Apoptosis assay (TUNEL immunostaining)
Follicular cell apoptosis was evaluated using the In Situ Cell Death Detection Kit-POD (Roche). After dewaxing and rehydration, the sections were treated with proteinase K and immersed in 3% H2O2. TUNEL working solution (label+enzyme) was applied for 60 minutes at 37 °C in the dark, followed by DAB staining. Primordial follicles were classified as apoptotic if their oocytes were TUNEL-positive. A paraffin-embedded ovarian section treated with DNase I (Takara Bio Inc.) served as the positive control.
9. Ovarian follicle classification and counting
Primordial follicles: oocytes surrounded by a single layer of squamous granulosa cells; Primary follicles: oocytes surrounded by a single layer of cuboidal granulosa cells; Secondary follicles: oocytes surrounded by two or more layers of cuboidal granulosa cells without an antrum; Antral follicles: follicles with an antral cavity.
To avoid duplication, only follicles containing an oocyte were counted. The primary to primordial follicle ratio was calculated by dividing the total number of primary follicles by the total number of primordial follicles in each group.
To assess the effects of ovarian tissue cryopreservation and thawing, follicle number was the secondary endpoint. Follicle density was used to evaluate the remaining follicle pool and was determined by counting follicles in a single central cross-section of each ovary stained with hematoxylin and eosin (H&E), divided by the cross-sectional area of that ovary.
In the ovarian tissue cryopreservation, thawing, and culture experiments, the primary endpoint was the total follicle count. Hence, the entire ovary was serially sectioned (5 µm thick, every 25 µm) and stained with H&E, and all follicles in all sections were counted. The sum was recorded as the total number of follicles per ovary (Figure 2).
10. Statistical analysis
The results were analyzed using JMP ver. 17 (JMP Statistical Discovery LLC). One-way analysis of variance was employed for comparisons among three groups, followed by a post hoc Tukey test if an overall significant difference was identified. The Mann-Whitney U test was utilized for comparisons between two groups. The results are presented as median (interquartile range [IQR]). p-values less than 0.05 were considered to indicate statistical significance.
Results
1. Vitrification activates mTOR—but not apoptosis—in primordial follicles, and rapamycin blocks this activation
Mouse ovaries were randomly assigned to three groups: no cryopreservation (fresh-control), cryopreservation/thawing without rapamycin and no subsequent culture (rapamycin-free non-culture), and cryopreservation/thawing with 750 nM rapamycin and no subsequent culture (rapamycin-treated non-culture). Primordial follicles were predominantly observed in the ovarian cortex. Immunostaining for pS6K revealed no pS6K-positive primordial follicles in the fresh-control group, whereas the rapamycin-free non-culture group exhibited intense pS6K immunoreactivity. In contrast, the rapamycin-treated group displayed only occasional pS6K-positive primordial follicles. The median pS6K-positive rate for primordial follicles in the fresh-control versus rapamycin-free versus rapamycin-treated groups was 7.1 (IQR, 5.9 to 8.5) vs. 87.9 (IQR, 83.0 to 89.8) vs. 19.0 (IQR, 17.1 to 19.2), respectively. The pS6K-positive rate differed significantly different between the rapamycin-free and fresh-control groups (p<0.001) and between the rapamycin-free and rapamycin-treated groups (p<0.001). TUNEL staining indicated the absence of apoptotic primordial follicles in all groups. Moreover, no significant differences were observed in the number of primordial follicles per unit area among the three groups, with medians of 12.6 (IQR, 11.7 to 14.0) for the fresh-control, 13.7 (IQR, 13.5 to 13.8) for the rapamycin-free, and 13.5 (IQR, 12.7 to 14.1) for the rapamycin-treated group. Thus, although closed vitrification and thawing activated the mTOR pathway in primordial follicles without causing apoptosis, adding rapamycin to the cryoprotectant solution successfully inhibited this activation. However, primordial follicle numbers were similar among the three groups (Figure 3).
2. After cryopreservation, thawing, and culture, excessive follicular activation through the mTOR pathway leads to primordial follicle loss, which is ameliorated by rapamycin
Although short-term cryopreservation and thawing activated mTOR in primordial follicles, it remained unclear whether activated primordial follicles subsequently developed into growing follicles. Therefore, a 5-day organ culture was performed after cryopreservation and thawing. Two groups were compared: the rapamycin-free culture group (no rapamycin added) and the rapamycin-treated culture group (750 nM rapamycin added to both the cryopreservation and culture media).
Low-magnification H&E staining showed no distinct differences in stromal tissue between the two groups. However, most primordial follicles in the rapamycin-free culture group exhibited intense pS6K immunoreactivity, whereas those in the rapamycin-treated culture group showed no pS6K signal. Furthermore, significant differences were observed in primordial follicle numbers and the ratio of primary to primordial follicles. In the rapamycin-free versus the rapamycin-treated culture group, the number of primordial follicles was 289 (IQR, 241 to 292) vs. 605 (IQR, 448 to 704) (p=0.03), respectively; the number of primary follicles was 69 (IQR, 69 to 70) vs. 54 (IQR, 37 to 58) (p=0.27), that of secondary follicles was 88 (IQR, 65 to 99) vs. 58 (IQR, 47 to 65) (p=0.12), that of antral follicles was 135 (IQR, 78 to 154) vs. 118 (IQR, 101 to 128) (p=1.00), and the ratio of primary to primordial follicles was 0.24 (IQR, 0.17 to 0.29) vs. 0.08 (IQR, 0.07 to 0.11) (p=0.008) (Figure 4).
Discussion
This study yielded two notable findings. First, closed-system vitrification activated the mTOR pathway in follicles, and this activation was effectively suppressed by an mTOR inhibitor. Second, the addition of rapamycin during cryopreservation, thawing, and subsequent culture significantly inhibited the excessive activation of the mTOR pathway, thus preserving the pool of primordial follicles (Figure 5).
Regarding the technical aspects of vitrification, it is believed that the design of the container used in closed-system vitrification plays a critical role in cell survival during ovarian tissue freezing. Theoretically, the closed system provides high hermeticity and prevents cross-contamination; however, such containers could potentially impede rapid freezing. In clinical practice, commercially available closed-system ovarian tissue cryopreservation kits are placed on a metal plate with high thermal conductivity, sealed within a pouch, and then immersed in liquid nitrogen. This high thermal conductivity enables rapid freezing, and indeed, closed-system vitrification has been reported to achieve follicle survival rates comparable to those observed with slow freezing and open-system vitrification methods [8]. Nonetheless, our study demonstrated that even under these rapid freezing conditions, the mTOR pathway is activated, leading to primordial follicle loss, and that mTOR inhibitors can effectively counteract this activation.
A review of the literature on the use of mTOR inhibitors in ovarian tissue cryopreservation, thawing, and transplantation reveals the following insights. Celik et al. [19,20], using a rat model in two foundational studies employing either slow freezing or open-system vitrification of ovarian tissue, demonstrated that follicle loss is associated with decreased expression of tuberous sclerosis complex (TSC) components and phosphatase and tensin homolog (PTEN) together with activation of the mTOR pathway; furthermore, systemic administration of rapamycin for 7 days following ovarian transplantation can attenuate the loss of primordial follicles. Similarly, Sato and Kawamura [30] showed that, in immunodeficient mice transplanted with human ovarian cortical fragments, systemic rapamycin treatment for 4 weeks post-transplantation inhibited the activation and development of primordial follicles. Liu et al. [21] reported that adding rapamycin to the freezing medium during open‑system vitrification of mouse ovarian tissue mitigated immediate post‑thaw mTOR activation and preserved primordial follicle numbers after in vivo transplantation. Furthermore, research groups led by Bindels et al. [13,22] and Terren et al. [12] have published three studies using mouse models, covering freezing procedures, post‑thaw whole‑organ cultures, and in vivo transplantation. This research has demonstrated that the addition or systemic administration of rapamycin or phosphatidylinositol 3-kinase inhibitors improves primordial follicle survival and even yields a significant increase in offspring following transplantation [12,13,22].
In summary, it is established that mTOR inhibitors protect primordial follicles in both in vivo transplantation and whole-organ culture models, as well as during freezing and thawing processes employing open-system vitrification and slow freezing methods. However, a gap remains regarding whether the mTOR pathway is similarly activated during the cryopreservation and thawing of ovarian tissue using closed-system vitrification, and whether mTOR inhibitors can provide a protective effect in this context. As clinical studies have shown that ovarian tissue damage profiles vary by cryopreservation method, it is challenging to directly extrapolate data from one method to another [23,24]. Therefore, our findings—that closed‑system vitrification activates the mTOR pathway in follicles and that mTOR inhibition significantly preserves primordial follicles during subsequent whole‑organ culture—help bridge this gap when considered alongside previous studies. Continuous administration of mTOR inhibitors throughout cryopreservation, thawing, and transplantation may thus present a promising strategy for protecting primordial follicles in closed-system vitrification. The clinical implications of these results are substantial.
Ovarian follicle loss following cryopreservation and transplantation of ovarian tissue is primarily driven by ischemia-reperfusion injury, which induces aberrant activation of primordial follicles. Accordingly, current protective strategies include antioxidant agents (melatonin, N-acetylcysteine, resveratrol, erythropoietin), angiogenesis-promoting approaches (stem cells, angiogenic and growth factors), and direct inhibition of follicle activation (mTOR inhibitors and anti-Müllerian hormone) [10,11,31,32]. All of these have been investigated in preclinical models, but no head-to-head comparisons have yet been reported.
Against this background, a major advantage of using mTOR inhibitors is that rapamycin is already employed in assisted reproductive technologies (e.g., in vitro fertilization and embryo transfer) and has not been associated with significant teratogenic risk [32-34]. Another advantage relates to the typical patient population eligible for ovarian tissue cryopreservation—many are prepubertal girls or young women with hematologic malignancies or bone and soft-tissue sarcomas who may harbor occult ovarian micrometastases. mTOR inhibitors offer not only ovarian protection but also direct antitumor activity; indeed, at least one report has demonstrated that mTOR inhibition can completely eradicate Ewing sarcoma metastases from human ovarian cortical tissue fragments [35].
This study has several limitations. Guided by the ‘3R’ principles (replacement, reduction, and refinement), we used the minimum number of animals required to achieve statistical significance for our primary endpoints; consequently, confidence intervals are wide, and subtle effects may have been missed. We did not test in vivo transplantation of cryopreserved-thawed ovaries, nor did we include a group that received rapamycin only during cryopreservation and warming. Even so, earlier transplantation studies with fresh or slow‑frozen tissue show that mTOR activation drives follicle loss and that systemic mTOR inhibition mitigates this damage [20,30]; our work bridges those findings by focusing on the cryopreservation and warming phases. Whole‑organ culture, although widely used to assess folliculogenesis [36-42], remains a surrogate for in vivo physiology. Although follicle loss via excessive activation was demonstrated by the primary‑to‑primordial follicle ratio after 5 days of culture, post‑thaw morphological measures are insensitive to early activation events. Published data indicate that early activation markers peak approximately 24 hours after thawing [12]. Therefore, future studies should incorporate molecular readouts—such as forkhead box O3 (Foxo3a) nuclear export assessed around 24 hours post‑thaw—to capture these initial activation responses. Larger cohorts, multiple time points, and in vivo transplantation experiments will be required to confirm and extend these observations.
In conclusion, our study demonstrated that although closed‑system vitrification and thawing of ovarian tissue induce follicle loss by activating the mTOR pathway, rapamycin substantially contributes to the preservation of primordial follicles. These findings confirm that the ovarian protective effects of mTOR inhibitors—previously established in open-system vitrification, slow freezing, and transplantation models—also apply to the increasingly adopted closed-system vitrification method. Consequently, our results support the integration of rapamycin into cryopreservation protocols as a promising strategy for safeguarding the reproductive capacity of patients with cancer.
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