Improving Follicle Development After Cryopreservation of Prepubertal Ovarian Tissue Through In Vitro Activation

Improving Follicle Development After Cryopreservation of Prepubertal Ovarian Tissue Through In Vitro Activation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Improving Follicle Development After Cryopreservation of Prepubertal Ovarian Tissue Through In Vitro Activation Yang Jinze, Zhiyun Zhu, Mengmeng Zhang, Mengyuan Tian, Yu Wu, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8321884/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Fertility preservation in pediatric patients requires effective cryopreservation of ovarian tissue, yet the relative performance of slow freezing and vitrification in this age group remains unclear. This study compared the two cryopreservation methods in prepubertal ovarian tissue and examined their functional consequences through in vitro activation and short-term culture. Methods: Ovarian tissues from forty-six girls aged one to fourteen years were processed fresh, slow-frozen, or vitrified. Follicular morphology and developmental stage assessed by hematoxylin and eosin staining; apoptosis assessed by TUNEL assay; follicular activation assessed by Western blotting and immunohistochemistry; follicular growth after IVA culture. Appropriate statistical analyses were applied based on data distribution, including t-tests, non-parametric tests, and contingency table analyses. Results: Slow freezing preserved follicular morphology better than vitrification, with significantly less oocyte damage and stromal cell apoptosis. IVA significantly increased pFOXO3a/FOXO3a ratios at day 2 in both groups and promoted nuclear translocation of pFOXO3a. By day 6, IVA-treated tissues showed a higher increase in primary follicle numbers compared to untreated controls, particularly in the slow freezing group. Conclusions: Slow freezing is more effective than vitrification for pediatric ovarian tissue cryopreservation. IVA further enhances follicular activation after thawing and may provide a promising strategy for optimizing pediatric fertility preservation. Pediatric ovarian tissue cryopreservation slow freezing vitrification in vitro activation fertility preservation Figures Figure 1 Figure 2 Figure 3 Figure 4 Backgrounds The disease-free survival rate of children with malignant tumors has increased with the continuous development of cancer treatments ( 1 , 2 ). Non-surgically induced premature ovarian insufficiency occurred in 9% of children with cured malignant tumors ( 3 ), highlighting the necessity of fertility preservation for these children. Ovarian tissue cryopreservation (OTC) and transplantation (OTT), are the indispensable methods of fertility preservation for children and patients whose reproductive toxic therapy cannot be delayed ( 4 – 6 ). To date, more than 189 live births hitherto have been reported ( 7 ), with some scholars estimating the number exceeds 300 cases ( 8 ). Most of the live birth cases were obtained through slow freezing, while vitrification is a new technology that resulted in 5 live births ( 9 , 10 ). The ovaries of children differ structurally and developmentally from women of childbearing age. The ovarian follicle density of children ranges from 3,281.1 to 16,414.9 follicles/mm3, which is significantly higher than that of adults (53.3 to 733.7 follicles/mm³)( 11 ). In children, there is a spontaneously activated population of follicles in the ovaries that gradually undergoes atresia, namely "the first wave of follicles," while a large number of primordial follicles in the cortex remain in a dormant state, which can be activated during reproductive period, called "adult wave of follicles" ( 12 , 13 ). There is still a lack of research analyzing the impact of follicle distribution with different developmental potentials on the fertility preservation of children. Phosphatase and tensin homologs (PTEN) and phosphatidylinositol-3 kinase (PI3K) are crucial pathways to regulate follicle development, and the role of PTEN/PI3K/FOXO3 in activating primordial follicles of rodents has been fully elucidated ( 14 ). An in vitro activation (IVA) protocol has been developed to activate dormant follicles in the ovarian cortex of mouse by using PTEN inhibitors and PI3K activators ( 15 , 16 ). Clinically, IVA has been mainly applied in adult patients with diminished ovarian reserve, with several cases of live birth reported ( 17 ). Despite IVA has been combined with OTC and transplantation in some studies ( 18 ), there have been no reported cases of successful IVA of ovarian tissue from the prepubertal patients. Therefore, the purpose of this study is to 1) understand the impact of the particularity of ovarian tissue structure of children on the fertility preservation by analyzing the morphological characteristics of ovarian tissue, 2) investigate the survival of ovarian tissues after vitrification and slow freezing, and 3) determine whether the inhibition of PTEN and activation of PI3K can activate follicles of children in vitro through an in vitro culture system. Methods Study participants and ethical approval Use of human ovarian tissue was approved by Peking University Third Hospital Medical Science Research Ethics Committee (reference 2019SZ031). The study population included a total of 46 female children aged 1 to 14 years undergoing ovarian-sparing local mass excision from January 2019 to March 2025. The diagnosis of the patients included teratoma, ovarian cyst or other benign ovarian diseases. Ovarian tissue from 4 adult women was used as the controls. All patients or their guardians had provided written informed consent to donate their ovarian tissue to research. Slow freezing and thawing The slow freezing and thawing procedure was performed according to the method of Donnez et al ( 19 ). with a slight modification. Briefly, ovarian pieces were first equilibrated in freezing media, then individually transferred into cryovials containing 1.8 ml freezing media at 4°C. The cryovials were cooled in a programmable freezer (Kyro 360 − 1.7, Planer Inc., UK). For thawing, the cryovials were immersed in a water bath at 37°C with gentle shaking. When the liquid in the cryovials melted, tissues were transferred into thawing media. Vitrification and thawing The vitrification procedure was performed according to the method of Suzuki et al ( 20 ). with slight modifications. Ovarian pieces were vitrified using a two-step exposure to equilibration and vitrification solutions and then plunged into liquid nitrogen for storage. For thawing, samples were transferred to thawing media. Ovarian tissue culture After thawing, ovarian tissues were cultured in 6-well plate by adding 4mL media. The culture media was as reported by wang( 21 ) et al. All tissues were cultured at 37℃ under 5% CO 2 for up to 6 days. Every 2 days, half of the culture medium was replaced. In vitro activation of primordial follicle Ovarian tissues of IVA group were treated with 30µM bpv (MedChemExpress, HY-128693) and 150 µg/mL 740Y-P (MedChemExpress, HY-P0175) in culture media for 24h and only 150µg/mL 740Y-P for next 24h. For the control group, the culture media were replaced at the same time as the IVA group. Ovarian tissues were collected on day 2 of culture to evaluate the efficiency of follicular activation following IVA. To assess the subsequent developmental potential of activated follicles, tissues were harvested on day 6 of culture. Follicle number Counting Ovarian tissues were collected and fixed in 10% neutral buffered formalin fixative for 20 hours, then embedded in paraffin. Follicle quantification was performed following the protocol established by Yano Maher et al.( 22 ). Briefly, the paraffin blocks were cut into serial sections of 5 µm. Every 5th section was stained with hematoxylin and eosin (H.E.) for follicle classification and counting. Count the total number of follicles, and divide the number of follicles at each stage by the total number of follicles to obtain the distribution of follicles, expressed as a percentage. The primary follicle increasing rate is expressed as the difference between the proportion of primary follicles among total follicles after culture and that before culture, divided by the proportion before culture, and presented as a percentage. This parameter reflects the rate of increase in the proportion of primary follicles during the culture period, serving as an indicator of follicular activation efficiency. Detection and quantification of apoptosis For TUNEL technology, tissue sections were dewaxed and rehydrated. Tissue sections were incubated for 1h with In Situ Cell Death Detection Kit, TMR Red (Roche, 12156792910, Germany), at 37℃ in a humidified atmosphere in the dark. Samples were analyzed by Opereta CLS (PerkinElmer, USA). In the same batch, prepare a positive control (by treating the sample with DNase I for 10 minutes at 37°C) and a negative control (by incubating only with the lable solution without adding TdT enzyme). The positive cell rate in the tissue is defined as the number of positive cells divided by the total number of cells, expressed as a percentage. Immunoblotting analysis Ovarian proteins were extracted by Radio Immunoprecipitation Assay (RIPA) lysis buffer (ThermoFisher, 89901), containing Protease and Phosphatase Inhibitor Cocktail (ThermoFisher, 78442). A total of 20 µg proteins in each sample were loaded and separated by electrophoresis (Bio-Rad, USA, 1610732). After electronic transfer (Bio-Rad, 1610734), the Nitrocellulose Membrane (Millipore, Germany, HATF08130) were blocked in 5% blocking buffer (Bio-Rad, 12010020) for 10min and then incubated for 14h at 4°C with specific antibodies. Antibodies included p-FOXO3a (1:1000; Abcam, UK, ab47285), FOXO3a (1:2000, HUABIO, China, ER1706-79), and β-actin (1:2000, Cell Signaling Technology, USA, 4970). After washing with Tris Buffered Saline with Tween 20 (TBST) for three times, the Horseradish Peroxidase (HRP)-conjugated relative secondary antibodies were then used to detect proteins through enhanced chemiluminescence on the Tanon 5200 analysis system. Immunohistochemistry After deparaffinization and rehydration, incubate the sections with 3% hydrogen peroxide diluted in methanol for 10 minutes at room temperature. Antigen retrieval was then carried out by heating the sections in 10 mM citrate buffer (pH 6.0). The tissue sections were subsequently incubated overnight at 4°C with primary antibodies p-FOXO3a (1:100). Sections were incubated with HRP-conjugated secondary antibodies and visualized using a DAB detection system. Follicles were classified based on the nuclear translocation of pFOXO3a: follicles showing nuclear localization of pFOXO3a were considered positive, while those without nuclear translocation were classified as negative. Statistical analysis Statistical analysis was performed using GraphPad Prism version 8.0 (GraphPad Software, USA). For all analyses, data were first assessed for normality and homogeneity of variance. For datasets that met the assumptions of normal distribution and equal variance, comparisons between two groups were conducted using the unpaired or paired Student’s t -test, depending on whether the samples were independent or matched (derived from the same patient). Comparisons among three or more groups were performed using one-way analysis of variance (ANOVA). For non-normally distributed or heteroscedastic data, the Mann–Whitney U test was applied. Categorical (binary) outcomes were analyzed using contingency table (chi-square or Fisher’s exact) tests. A significance threshold of α = 0.05 was adopted. Where applicable, 95% confidence intervals (CIs) and statistical power (effect size, post hoc power) were reported to support the robustness of the findings. Results Follicular Composition and Abundance in Prepubertal Ovarian Tissue To characterize the follicular composition of prepubertal ovarian tissue, we examined the distribution of follicle stages in freshly collected samples from forty-six girls. All the children had not menstruated. The patients’ characteristics are reported in Table 1 . The distribution of pediatric ovarian tissue samples across the experimental procedures is outlined in the flow diagram in Fig. 1. Antral follicles were observed in all age groups (Fig. 2B, Figure S1 ). Primordial and primary follicles were the main types of ovarian follicles in children of all ages (Fig. 2A). The proportions of primordial follicles in the low ( 9 years old) age groups of children's ovaries were 68.96% ± 19.50%, 65.17% ± 19.09%, and 54.13% ± 20.50% (F = 2.08, p = 0.115, effect size η 2 = 0.12, 95%CI of η 2 : 0.04–0.34). The proportions of primary follicles in the low, medium, and high age groups of children's ovaries were 26.73% ± 13.31%, 36.34% ± 25.10%, and 39.80% ± 15.69% (F = 1.88, p = 0.15, effect size η 2 = 0.11, 95%CI of η 2 : 0.05–0.31). These findings demonstrate that prepubertal ovarian tissue contains a rich follicular reserve dominated by primordial and primary follicles, suggesting that it represents an excellent substrate for activation and fertility preservation strategies. Figure 1. Flow diagram of sample allocation and experimental procedures. This flow chart summarizes the allocation of ovarian tissue samples obtained from forty-six prepubertal girls undergoing ovarian-sparing surgery. All samples were first evaluated by fresh tissue morphology analysis. Following screening, tissues were assigned to cryopreservation by slow freezing or vitrification. Three ovarian tissue samples were too small to be divided into experimental fragments and therefore were not subjected to cryopreservation procedures. Five patients contributed matched samples for apoptosis assessment under fresh, slow-frozen, and vitrified conditions. Ten patients provided ovarian tissue for six-day in vitro culture to compare post-thaw developmental potential. Molecular activation analyses (pFOXO3a immunohistochemistry and Western blotting) were performed on thawed tissues derived from six donors involved in the activation experiments. In vitro activation experiments included nine patients in the slow freezing group and thirteen patients in the vitrification group. Figure 2. Fresh ovarian tissue histology in children of different ages. (A) Distribution of follicles in low-aged (0 ~ 3 years old, n = 6), middle-aged (4 ~ 8 years old, n = 18), and high-aged (over 9 years old, n = 22) children ovaries. Data are shown as mean ± SEM. Pr, Primordial follicle; P, Primary follicle; S, Secondary follicle; A, Antral follicle. (B) Ovarian histology shown by hematoxylin and eosin staining of different ages and stages. Representative images of primordial follicles, primary follicles, secondary follicles, antral follicles were displayed. Except for those directly marked in the figure, all scale bars represent 50µm. The black arrows indicate the primordial follicles while the white arrows indicate the primary follicles. Figure S1 Fresh ovarian tissue histology in Adults. Ovarian histology shown by hematoxylin and eosin staining. Representative images of primordial follicles, primary follicles, secondary follicles, antral follicles were displayed. Except for those directly marked in the figure, all scale bars represent 50µm. The black arrows indicate the primordial follicles while the white arrows indicate the primary follicles. Table 1 Population characteristics low-age group n = 6 middle-age group n = 18 high-age group n = 22 p -value age (years) 1.8 (0.9 to 2.1) 5.8 (4.8 to 7.5) 11.0 (11.0 to 14.0) weight(kg) 11.6 (10.0 to 14.0) 21.5 (18.0 to 25.1) 49.0 (33.0 to 62.5) height(m) 0.79 (0.72 to 0.89) 1.22 (1.18 to 1.29) 1.58 (1.47 to 1.67) BMI 16.57 (15.77–18.76) 16.30 (14.12 to 16.80) 20.59 (16.84 to 24.88) Diagnosis Mature teratoma 3 16 16 Ovarian cyst 3 2 4 Teratoma comorbid with cyst 0 0 2 Reproductive endocrine hormone FSH(IU/L) 0.925 (0.100 to 2.430) 0.955 (0.585 to 1.620) 3.170 (2.150 to 6.190) p = 0.0198 LH(IU/L) 0.10 (0.10 to 0.18) 0.10 (0.10 to 0.13) 1.39 (0.33 to 3.55) p = 0.0084 E 2 (pg/mL) 60.0 (42.3 to 199.0) 25.5 ( 13.8 to 54.3) 50.0 (17.56 to 126.0) p = 0.5976 P(ng/mL) 0.64 (0.40 to 1.10) 0.68 (0.43 to 2.00) 0.91 (0.43 to 1.30) p = 0.1932 T(ng/mL) 0.69 (0.69 to 2.36) 0.69 (0.69 to 3.20) 1.88 (0.69 to 14.8) p = 0.0566 PRL(ng/mL) 26.80 (7.31 to 50.88) 17.15 (7.13 to 25.85) 16.33 (10.14 to 37.48) p = 0.5620 AMH(ng/mL) 2.71 (1.47 to 3.31) 3.16 (1.31 to 5.12) 4.49 (2.88 to 6.01) p = 0.0573 Data are shown as IQR [Median (Q1 to Q3)]. FSH: follicle-stimulating hormone; LH: luteinizing hormone; E2: estradiol; P: progesterone hormone; T: testosterone; PRL: prolactin; AMH: anti-mullerian hormone The distribution and morphology of different development stages of ovarian tissue after cryopreservation/thawing by different methods To compare the tissue viability following slow freezing and vitrification, apoptotic levels were analyzed in ovarian tissues from five patients under three conditions: fresh, slow freezing, and vitrification. The proportions of apoptotic cells in the three groups were 2.32%±2.70%, 13.91%±3.66%, and 49.91%±14.20%, showing a statistically significant difference ( p = 0.0002). Compared with fresh ovarian tissue, both slow freezing and vitrification significantly increased apoptosis (Fresh vs. Sf: p = 0.0049, 95%CI: −16.3, − 6.8, Cohen’s d = 3.03; Fresh vs. Vit: p = 0.0054, 95%CI: −65.5-−29.7, Cohen’s d = 3.30). In addition, vitrification resulted in markedly higher apoptosis than slow freezing ( p = 0.0042, 95%CI: −53.1-−18.9, Cohen’s d = 2.62). Meanwhile, staining results indicated that apoptotic cells were distributed in both follicles and stromal cells (Fig. 3A, E). To further assess follicular damage following different cryopreservation methods, the survival status of 311 follicles was analyzed using contingency table analysis. The proportions of follicular damage in the fresh, slow-freezing, and vitrification groups were 6.73%, 26.09%, and 59.65% (Fig. 3B, D). There is evidence indicating that slow freezing is associated with abnormal follicular morphology ( p = 0.0004, 95%CI: 1.097–1.565, relative risk RR = 1.262). There is also evidence of an association between vitrification and follicular damage ( p < 0.0001, 95% CI: 1.745–3.265, relative risk RR = 2.331). Additionally, the proportion of damaged follicles was significantly higher after vitrification compared to slow freezing ( p = 0.0008, 95% CI: 1.294–2.667, relative risk RR = 1.832). The proportion of primordial follicles in ovarian tissue after thawing was 68.16% ± 15.73% and 68.26% ± 8.96% for slow freezing and vitrification methods, which was not significantly different from the fresh group (59.90% ± 27.68%) ( p = 0.3800, effect size η 2 = 0.079, 95%CI of η 2 : 0.022–0.311,). The proportion of primary follicles in ovarian tissue after thawing was 31.12% ± 15.11% and 30.29% ± 6.91% for slow freezing and vitrification methods, which was not significantly different from the fresh group (38.09% ± 26.57%) ( p = 0.82, effect size η 2 = 0.05, 95%CI of η 2 : 0.006–0.843). (Fig. 3C). These findings indicate that slow freezing causes less ovarian tissue damage compared to vitrification. Furthermore, to evaluate the follicular developmental potential of ovarian tissue following slow freezing and vitrification, ovarian samples from ten patients were subjected to a 6-day in vitro culture. The increase rate of primary follicles in the slow freezing group was 14.41% ± 7.51%, which was significantly higher than 5.13% ± 5.16% in the vitrification group (U = 22.0, p = 0.038, 95%CI: 0.026–0.164, Cohen’s d = 1.39) (Table S1 ). These findings indicate that ovarian tissue subjected to slow freezing exhibits greater developmental potential during in vitro culture compared to vitrified tissue. Figure 3. Apoptotic and morphological assessment of prepubertal ovarian tissue following fresh processing, slow-freezing and vitrification. (A) Quantification of apoptotic cells by TUNEL assay in fresh (Fresh), slow-freezing (Sf) and vitrification (Vit) groups. Bars show mean ± SEM; Fresh vs. Sf, p = 0.0049; Fresh vs. Vit, p = 0.0054; Sf vs. Vit, p = 0.0042. (B) Proportion of morphologically normal (black) and damaged (gray) follicles after each treatment. Cryopreservation significantly increased follicular damage (χ² test, p < 0.0001).(C) Distribution of follicle developmental stages— primordial (Pr), primary (P), secondary (S) and antral (A)—in each group. No marked shifts in stage composition were observed following either slow‐freezing or vitrification.(D) Representative hematoxylin & eosin–stained sections of ovarian cortex after processing under each condition. Scale bar = 50 µm.(E) Representative fluorescence images of TUNEL assay showing nuclei (DAPI, blue) and apoptotic cells (TUNEL, yellow). Scale bar = 100 µm. IVA enhances follicular activation and early development in thawed prepubertal ovarian tissue Using the PTEN inhibitor bpv and PI3K activator 740Y-P, IVA can activate primordial follicles from frozen-thawed pediatric ovarian tissues, offering a promising strategy for preserving fertility in children. Due to the presence of spontaneously activated first-wave follicles in pediatric ovarian tissue, it remains unclear whether the adult IVA protocol is capable of activating primordial follicles in pediatric ovarian tissue. To address this question, pediatric ovarian tissues subjected to different cryopreservation methods were treated with IVA under a defined culture system. To confirm molecular changes associated with activation, Western blot analysis was performed to quantify the ratio of phosphorylated FOXO3a to total FOXO3a (pF/F). The slow freezing IVA group showed a significantly higher pF/F compared to the control group ( p = 0.0078, mean difference: 0.68, 95%CI: -1.04 to -0.32, Cohen’s d = 4.65), as well as the vitrification group ( p = 0.002, mean difference: 1.75, 95%CI: -2.24 to -1.25, Cohen’s d = 9.73) (Fig. 4C-3F). These findings suggest that the activation protocol using bpV/740Y-P is effective in activating the primordial follicles of cryopreserved pediatric ovarian tissue. To evaluate follicular activation following IVA, ovarian tissues were first analyzed for pFOXO3a expression and localization by day 2 of culture (Fig. 4A-3B). In the slow freezing group, a total of 142 follicles were analyzed. Following IVA, 64.89% of follicles exhibited nuclear translocation of pFOXO3a, compared to 25.00% in the control group, indicating that there is evidence of an association between IVA and pFOXO3a nuclear translocation ( p < 0.0001, 95%CI: 0.2257–0.6162, relative risk RR = 2.596). Similarly, in the vitrification group, 279 follicles were included, with 79.25% of follicles showing nuclear translocation after IVA, compared to 39.58% in the control group, suggesting that IVA significantly enhances pFOXO3a nuclear translocation during post-vitrification culture ( p < 0.0001, 95%CI: 0.3975–0.6166, relative risk RR = 2.002). Furthermore, contingency table analysis was performed to compare the proportion of follicles exhibiting nuclear translocation after IVA between slow-frozen and vitrified ovarian tissues. The results demonstrated that the incidence of nuclear translocation was significantly higher following slow freezing compared to vitrification ( p = 0.022, 95%CI: 1.038–1.470, relative risk RR = 1.221). To further investigate the follicular development potential of cryopreserved ovarian tissue following IVA, ovarian culture outcomes were analyzed from nine patients in the slow-freezing group and thirteen patients in the vitrification group. Representative morphological features of the cultured tissues are shown in Fig. 4G, H. In slow freezing group, IVA significantly increased primary follicle increasing rate compared to control group (47.09%±19.36% vs. 22.60%±5.97%, p = 0.031, 95%CI: 0.10–1.52, Cohen’s d = 4.13). Similarly, in vitrification group, IVA significantly increased primary follicle increasing compared to control group (65.93%±64.25% vs. 23.58%±67.26%, p = 0.005, 95%CI: 0.06–0.82, Cohen’s d = 3.71). Further analysis of the proportions of follicles at each developmental stage is shown in Fig. 4I, 4J. In the slow freezing group, the proportion of primordial follicles was significantly reduced in the activation group ( p = 0.0005, 95%CI: 0.1072–0.3930), while the proportion of primary follicles was significantly higher in the activation group compared to the control ( p = 0.0012, 95%CI: -0.3713- -0.0855). In contrast, there is no statistically significant differences were observed between the activation and control groups in terms of follicular stage distribution in vitrification group. These results indicate that IVA promotes follicular development during culture, but there is no evidence of difference between the two cryopreservation methods. Figure 4. IVA promotes the activation of primordial follicles in children's ovaries after cryopreservation-thawing. (A) H.E. staining of ovarian cortical sections from vitrified (Vit) and slow-frozen (Sf) tissues, either without activation (NC) or following IVA. (B) Immunohistochemical detection of phosphorylated FOXO3a (pFOXO3a) in the same four groups. Scale bars, 50 µm. (C, D) Quantification of pFOXO3a‐positive (black) versus pFOXO3a‐negative (gray) follicles in Vit and Sf groups. IVA markedly increased the proportion of pFOXO3a‐positive follicles in both Vit and Sf tissues (p < 0.0001, χ² test). (E, F) Representative Western blots for pFOXO3a, total FOXO3a and β‐actin in NC versus IVA samples of Vit and Sf tissues. Densitometric analysis of the pFOXO3a/FOXO3a ratio, showing significant elevation of FOXO3a phosphorylation after IVA in both Vit (p = 0.0140) and Sf (p = 0.0357) groups (Student’s t‐test). (G) H.E. stained sections of vitrified (Vit) ovarian cortex in fresh (immediately fixed), non-activated control (NC) and IVA groups. Right: percentage increase in primary follicles following IVA (p = 0.0286). (H) H.E. stained for slow-frozen (Sf), showing a similar IVA- induced rise in primary follicles (p = 0.0331). Scale bars, 50 µm. (I) Corresponding follicle distribution for Vit tissues, revealing no significant difference in stage proportions after IVA. (J) Follicle-stage distribution in Sf tissues: bar graph of mean ± SD percentages of primordial, primary and secondary follicles in NC (black) versus IVA (gray) groups; IVA significantly reduced primordial (p = 0.0005) and increased primary (p = 0.0012) follicle proportions. Figure S2 . Representative Western blot membranes for the vitrification (Vit) and slow-freezing (Sf) groups. Each membrane shows protein expression in negative control (NC) and in vitro activation (IVA) samples (N = 3). For each group, the original membrane was cut horizontally at the midline to allow incubation with different primary antibodies. Discussion In this study, we described the distribution of follicles at all stages in fresh ovarian tissue from children, investigated the effectiveness of slow freezing and vitrification on child ovarian tissues, and examined the applicability of PTEN/PI3K IVA to primordial follicles of pediatric ovarian tissues. The results indicate that follicles at various developmental stages are present in the cortex of pediatric ovarian tissue. Slow freezing causes less damage to the ovarian tissue compared to vitrification. Moreover, the current adult IVA protocol is capable of activating primordial follicles in prepubertal ovarian tissue. Based on available reports, this study appears to be among the more extensive analyses of fresh ovarian tissue morphology in prepubertal patients. Previous studies on the proportion of follicles at various stages in ovaries of children have been hindered due to the difficulty in obtaining samples ( 11 , 23 – 26 ), and the medulla of the underaged ovarian tissue contains a large number of activated first-wave follicles ( 12 , 27 ). In addition, we found that there were antral follicles in the ovaries of 1-year-old children. This provided new insights into the developmental potential of the first wave of follicles, suggesting that the follicles of early children have the potency to develop into antral follicles. Although the fertility of the anthropic first wave of follicles has been controversial, studies in mice suggest that these follicles have the ability to produce embryos through in vitro fertilization and could be targeted for human fertility preservation ( 12 ). Overall, the abundance of follicles in prepubertal ovarian tissue indicates a favorable baseline for activation and offers a compelling rationale for prioritizing tailored preservation strategies in young patients. The efficacy of slow freezing and vitrification remains contentious. In this study, we reported a lower level of damaged follicles of slow frozen ovarian tissue of children than that of the vitrificated group after thawing, which was consistent with the findings of Dolmans et al. based on adult ovarian tissue( 28 , 29 ), indicating the advantageous preservative capacity of slow freezing in follicle viability. Recently, the vital role of the stromal cell in follicle growth and development has been gradually recognized ( 30 – 32 ). Our study found that both vitrification and slow freezing led to increased levels of apoptosis of stromal cells compared to fresh tissue, with vitrification inducing a higher degree of apoptosis than slow freezing. Prolonged in vitro culture may contribute to reduced follicular developmental competence( 33 ). We speculate that damage to stromal cells may be associated with the reduced in vitro developmental potential of follicles in vitrified-thawed ovarian tissue. Some studies have discussed vitrification as an alternative to slow freezing ( 34 ). However, evidence derived from adults should be cautiously extrapolated to children, especially considering that pediatric ovarian tissue preservation usually requires longer storage periods than adults. We recommend that capable centers should offer both cryopreservation methods concurrently for pediatric patients, taking into account the best interests of them. IVA can activate primordial follicles. However, current study has not explored whether IVA can alter the fate of "the first wave" follicles destined for apoptotic atresia ( 35 – 37 ). Nuclear translocation of pFOXO3a, the increased pF/F ratio, and the increased proportion of primary follicles during in vitro culture collectively support the efficacy of IVA in pediatric ovarian tissue. These findings suggest that despite the presence of the naturally occurring "first-wave" follicle activation in pediatric ovaries, ( 38 , 39 ), the follicles remain responsive to IVA, indicating that this approach may have applicability and practical value in pediatric ovarian tissue. Our study did not further investigate individual differences in follicular response to IVA across various age groups. Future studies could explore stratified activation strategies. In this study, follicle density was not reported because the ovarian tissues were obtained adjacent to benign lesions, and the amount of remaining healthy cortex varied considerably among patients. In several samples, the follicle density was extremely low due to limited residual tissue rather than true biological differences. Including these values would have introduced substantial variability and made the data difficult to interpret. Instead, we focused on the proportion of follicles at each developmental stage, which provides a more reliable indicator of follicular activation and developmental shifts in this cohort. Meanwhile, this study was limited to in vitro culture experiments, and some studies( 40 ) pointed out that the transplantation process is another essential factor limiting the live birth rate of OTC, so further in vivo studies are needed to explore the optimal scheme of child fertility preservation. In summary, the abundant early follicles and clear activation potential of prepubertal ovarian tissue highlight the opportunity—and necessity—to refine cryopreservation and activation strategies specifically for pediatric fertility preservation. IVA serves as an important complementary strategy for fertility preservation in pediatric patients and should be considered in clinical practice. The significance of this study lies in providing new technological strategies for fertility preservation in pediatric patients. By combining multiple techniques, it is possible to achieve optimal protection and promotion of fertility in children. Declarations Ethics approval and consent to participate Use of human ovarian tissue was approved by Peking University Third Hospital Medical Science Research Ethics Committee (reference 2019SZ031). All study procedures adhered to the ethical principles outlined in the Declaration of Helsinki and complied with the Administrative Measures for Ethical Review of Life Science and Medical Research Involving Humans Consent for publication Written informed consent for participation in the study and publication of anonymized data was obtained from all adult participants and from the parents or legal guardians of all pediatric participants. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests Funding National Natural Science Foundation of China (T2293764), National Key R&D Program of China (2022YFC2703000, 2022YFC2704400, 2022YFC2704401), and National Natural Science Foundation of China (82288102, 82171630). Authors' contributions J.Y., Z.Z., M.Z. and M.T. contributed equally as first authors and HY.W., H.W. and J.Y. contributed equally as co-corresponding authors to the paper. All authors contributed to manuscript editing and read and approved the final manuscript. J.Y., Z.Z. and J.Y. conceived the project; HY.W., H.W., J.Q. and J.Y. supervised the study; M.Z. and J.Y. were responsible for the data curation. M.Z., M.T., J.H., T.L., J.H. and J.Y. performed the experiments; Z.Z., W.Y., H.C., S.Y., X.S., Y.W. and H.W performed the operations. J.Y., M.Z., M.T. and J.H. analyzed most experiments and compiled the figures. J.Y. and Z.Z. wrote the manuscript with edits from J.Y. and J.Q. HY.W, J.Q., J.Y., and Y.W. contributed to obtaining funding. Acknowledgement We want to thank Dr. Zheng Wang for reviewing the English language of the article. We are grateful to Dr. Yan Liu (Peking University Health Science Center) for her guidance on follicular morphology. We are grateful to Jinghao Yan, Yu Lin, Saishuo Chang, Fuyi Zhu and the other colleagues in the operating theater (National Center for Children's Health) for their help in preserving and transporting clinical samples. We also express our appreciation to Jia Yang and Fengrui Yang (Peking University Health Science Center) for their work on follicular counting. References Smith MA, Altekruse SF, Adamson PC, Reaman GH, Seibel NL. 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Ayuandari S, Winkler-Crepaz K, Paulitsch M, Wagner C, Zavadil C, Manzl C, et al. Follicular growth after xenotransplantation of cryopreserved/thawed human ovarian tissue in SCID mice: dynamics and molecular aspects. Journal of Assisted Reproduction and Genetics. 2016;33(12):1585-93. Hopkins TIR, Bemmer VL, Franks S, Dunlop C, Hardy K, Dunlop IE. Micromechanical mapping of the intact ovary interior reveals contrasting mechanical roles for follicles and stroma. Biomaterials. 2021;277:121099. Kinnear HM, Tomaszewski CE, Chang FL, Moravek MB, Xu M, Padmanabhan V, et al. The ovarian stroma as a new frontier. Reproduction. 2020;160(3):R25-R39. Guo YC, Xue LR, Tang WC, Xiong JQ, Chen D, Dai Y, et al. Ovarian microenvironment: challenges and opportunities in protecting against chemotherapy-associated ovarian damage. Human Reproduction Update. 2024;30(5):614-47. Hossay C, Tramacere F, Camboni A, Cacciottola L, Van Kerk O, Donnez J, et al. P-437 Follicle activation in human ovarian tissue: impact of freezing, culture and grafting. Human Reproduction. 2022;37(Supplement_1):deac107.412. Shi QQ, Xie YD, Wang Y, Li SW. Vitrification versus slow freezing for human ovarian tissue cryopreservation: a systematic review and meta-anlaysis. Scientific Reports. 2017;7(1):8538. Hirshfield AN. Heterogeneity of Cell Populations that Contribute to the Formation of Primordial Follicles in Rats1. Biology of Reproduction. 1992;47(3):466-72. Ernst EH, Franks S, Hardy K, Villesen P, Lykke-Hartmann K. Granulosa cells from human primordial and primary follicles show differential global gene expression profiles. Human Reproduction. 2018;33(4):666-79. Rooda I, Hassan J, Hao J, Wagner M, Moussaud-Lamodière E, Jääger K, et al. In-depth analysis of transcriptomes in ovarian cortical follicles from children and adults reveals interfollicular heterogeneity. Nature Communications. 2024;15(1):6989. Albamonte MI, Calabro LY, Albamonte MS, Vitullo AD. FOXO3 and PTEN expression in the ovary of girls with extra-gonadal cancer with or without chemotherapy treatment prior to cryopreservation. Bmc Womens Health. 2023;23(1):509. Tarnawa ED, Baker MD, Aloisio GM, Carr BR, Castrillon DH. Gonadal expression of Foxo1, but not Foxo3, is conserved in diverse Mammalian species. Biol Reprod. 2013;88(4):103. Damous LL, Nakamuta JS, Soares-Jr JM, Maciel GAR, Simões RdS, Montero EFdS, et al. Females transplanted with ovaries subjected to hypoxic preconditioning show impair of ovarian function. Journal of Ovarian Research. 2014;7(1):34. Supplementary Files FigureS1.pdf Figure S1 Fresh ovarian tissue histology in Adults. Ovarian histology shown by hematoxylin and eosin staining. Representative images of primordial follicles, primary follicles, secondary follicles, antral follicles were displayed. Except for those directly marked in the figure, all scale bars represent 50μm. The black arrows indicate the primordial follicles while the white arrows indicate the primary follicles. FigureS2.pdf Figure S2. Representative Western blot membranes for the vitrification (Vit) and slow-freezing (Sf) groups. Each membrane shows protein expression in negative control (NC) and in vitro activation (IVA) samples (N = 3). For each group, the original membrane was cut horizontally at the midline to allow incubation with different primary antibodies. TableS1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8321884","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":590902155,"identity":"4a867ff3-802c-46e1-8d99-ee744c703c61","order_by":0,"name":"Yang 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01:29:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8321884/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8321884/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102991981,"identity":"a693a9e5-3f89-4931-a858-4c2be54dcb76","added_by":"auto","created_at":"2026-02-19 11:37:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":59492,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlow diagram of sample allocation and experimental procedures.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis flow chart summarizes the allocation of ovarian tissue samples obtained from forty-six prepubertal girls undergoing ovarian-sparing surgery. All samples were first evaluated by fresh tissue morphology analysis. Following screening, tissues were assigned to cryopreservation by slow freezing or vitrification. Three ovarian tissue samples were too small to be divided into experimental fragments and therefore were not subjected to cryopreservation procedures. Five patients contributed matched samples for apoptosis assessment under fresh, slow-frozen, and vitrified conditions. Ten patients provided ovarian tissue for six-day in vitro culture to compare post-thaw developmental potential. Molecular activation analyses (pFOXO3a immunohistochemistry and Western blotting) were performed on thawed tissues derived from six donors involved in the activation experiments. In vitro activation experiments included nine patients in the slow freezing group and thirteen patients in the vitrification group.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8321884/v1/4568a1677034745bc6a80c01.png"},{"id":102991987,"identity":"8bba6d60-187f-4184-8a31-6a217ec97c33","added_by":"auto","created_at":"2026-02-19 11:37:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":714384,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFresh ovarian tissue histology in children of different ages.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Distribution of follicles in low-aged (0~3 years old, n=6), middle-aged (4~8 years old, n=18) , and high-aged (over 9 years old, n=22) children ovaries. Data are shown as mean ± SEM. Pr, Primordial follicle; P, Primary follicle; S, Secondary follicle; A, Antral follicle. (B) Ovarian histology shown by hematoxylin and eosin staining of different ages and stages. Representative images of primordial follicles, primary follicles, secondary follicles, antral follicles were displayed. Except for those directly marked in the figure, all scale bars represent 50μm. The black arrows indicate the primordial follicles while the white arrows indicate the primary follicles.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8321884/v1/d9105026aaf56b42f1e041d2.png"},{"id":102991984,"identity":"33b792dc-680f-4bc0-8f28-238832bb9398","added_by":"auto","created_at":"2026-02-19 11:37:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":766253,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApoptotic and morphological assessment of prepubertal ovarian tissue following fresh processing, slow‐freezing and vitrification.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Quantification of apoptotic cells by TUNEL assay in fresh (Fresh), slow‐freezing (Sf) and vitrification (Vit) groups. Bars show mean ± SEM; Fresh vs. Sf, p = 0.0049; Fresh vs. Vit, p = 0.0054; Sf vs. Vit, p = 0.0042. (B) Proportion of morphologically normal (black) and damaged (gray) follicles after each treatment. Cryopreservation significantly increased follicular damage (χ² test, p \u0026lt; 0.0001).(C) Distribution of follicle developmental stages— primordial (Pr), primary (P), secondary (S) and antral (A)—in each group. No marked shifts in stage composition were observed following either slow‐freezing or vitrification.(D) Representative hematoxylin \u0026amp; eosin–stained sections of ovarian cortex after processing under each condition. Scale bar = 50 µm.(E) Representative fluorescence images of TUNEL assay showing nuclei (DAPI, blue) and apoptotic cells (TUNEL, yellow). Scale bar = 100 µm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8321884/v1/c421f9489e8c447a96356a48.png"},{"id":102991983,"identity":"2d7527f9-266a-4a53-82f3-71934f965f3c","added_by":"auto","created_at":"2026-02-19 11:37:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1700214,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIVA promotes the activation of primordial follicles in children's ovaries after cryopreservation-thawing.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) H.E. staining of ovarian cortical sections from vitrified (Vit) and slow‐frozen (Sf) tissues, either without activation (NC) or following IVA. (B) Immunohistochemical detection of phosphorylated FOXO3a (pFOXO3a) in the same four groups. Scale bars, 50 µm. (C, D) Quantification of pFOXO3a‐positive (black) versus pFOXO3a‐negative (gray) follicles in Vit and Sf groups. IVA markedly increased the proportion of pFOXO3a‐positive follicles in both Vit and Sf tissues (p \u0026lt; 0.0001, χ² test). (E, F) Representative Western blots for pFOXO3a, total FOXO3a and β‐actin in NC versus IVA samples of Vit and Sf tissues. Densitometric analysis of the pFOXO3a/FOXO3a ratio, showing significant elevation of FOXO3a phosphorylation after IVA in both Vit (p = 0.0140) and Sf (p = 0.0357) groups (Student’s t‐test). (G) H.E. stained sections of vitrified (Vit) ovarian cortex in fresh (immediately fixed), non-activated control (NC) and IVA groups. Right: percentage increase in primary follicles following IVA (p = 0.0286). (H) H.E. stained for slow-frozen (Sf), showing a similar IVA-\u003c/p\u003e\n\u003cp\u003einduced rise in primary follicles (p = 0.0331). Scale bars, 50 µm. (I) Corresponding follicle distribution for Vit tissues, revealing no significant difference in stage proportions after IVA. (J) Follicle‐stage distribution in Sf tissues: bar graph of mean ± SD percentages of primordial, primary and secondary follicles in NC (black) versus IVA (gray) groups; IVA significantly reduced primordial (p = 0.0005) and increased primary (p = 0.0012) follicle proportions.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8321884/v1/04c1e9b94d0cb240d951f0ac.png"},{"id":105728481,"identity":"48b319f7-f26f-4dee-9bba-7ce3cb5d9f67","added_by":"auto","created_at":"2026-03-30 11:11:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4718912,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8321884/v1/50d9486e-c5f1-461a-9477-e963eee994d5.pdf"},{"id":102991986,"identity":"341e2a90-aed2-4951-8e41-8eb561b9864a","added_by":"auto","created_at":"2026-02-19 11:37:33","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":126066,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1 Fresh ovarian tissue histology in Adults.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOvarian histology shown by hematoxylin and eosin staining. Representative images of primordial follicles, primary follicles, secondary follicles, antral follicles were displayed. Except for those directly marked in the figure, all scale bars represent 50μm. The black arrows indicate the primordial follicles while the white arrows indicate the primary follicles.\u003c/p\u003e","description":"","filename":"FigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8321884/v1/acdd34a8e0b5ebdb5ccd64d9.pdf"},{"id":102991982,"identity":"069ae9e4-fa95-4261-a32f-17df43ce2c94","added_by":"auto","created_at":"2026-02-19 11:37:32","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":83932,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S2. \u003c/strong\u003eRepresentative Western blot membranes for the vitrification (Vit) and slow-freezing (Sf) groups. Each membrane shows protein expression in negative control (NC) and in vitro activation (IVA) samples (N = 3). For each group, the original membrane was cut horizontally at the midline to allow incubation with different primary antibodies.\u003c/p\u003e","description":"","filename":"FigureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8321884/v1/7c54cb598712e2800a84da15.pdf"},{"id":102991985,"identity":"2f571568-0463-498f-b571-0ab4690473cc","added_by":"auto","created_at":"2026-02-19 11:37:33","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":20580,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8321884/v1/451f69356bd6ab7ea6dfc6e2.docx"}],"financialInterests":"","formattedTitle":"Improving Follicle Development After Cryopreservation of Prepubertal Ovarian Tissue Through In Vitro Activation","fulltext":[{"header":"Backgrounds","content":"\u003cp\u003eThe disease-free survival rate of children with malignant tumors has increased with the continuous development of cancer treatments (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Non-surgically induced premature ovarian insufficiency occurred in 9% of children with cured malignant tumors (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), highlighting the necessity of fertility preservation for these children. Ovarian tissue cryopreservation (OTC) and transplantation (OTT), are the indispensable methods of fertility preservation for children and patients whose reproductive toxic therapy cannot be delayed (\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). To date, more than 189 live births hitherto have been reported (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), with some scholars estimating the number exceeds 300 cases (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Most of the live birth cases were obtained through slow freezing, while vitrification is a new technology that resulted in 5 live births (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe ovaries of children differ structurally and developmentally from women of childbearing age. The ovarian follicle density of children ranges from 3,281.1 to 16,414.9 follicles/mm3, which is significantly higher than that of adults (53.3 to 733.7 follicles/mm\u0026sup3;)(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). In children, there is a spontaneously activated population of follicles in the ovaries that gradually undergoes atresia, namely \"the first wave of follicles,\" while a large number of primordial follicles in the cortex remain in a dormant state, which can be activated during reproductive period, called \"adult wave of follicles\" (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). There is still a lack of research analyzing the impact of follicle distribution with different developmental potentials on the fertility preservation of children.\u003c/p\u003e \u003cp\u003ePhosphatase and tensin homologs (PTEN) and phosphatidylinositol-3 kinase (PI3K) are crucial pathways to regulate follicle development, and the role of PTEN/PI3K/FOXO3 in activating primordial follicles of rodents has been fully elucidated (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). An in vitro activation (IVA) protocol has been developed to activate dormant follicles in the ovarian cortex of mouse by using PTEN inhibitors and PI3K activators (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Clinically, IVA has been mainly applied in adult patients with diminished ovarian reserve, with several cases of live birth reported (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Despite IVA has been combined with OTC and transplantation in some studies (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), there have been no reported cases of successful IVA of ovarian tissue from the prepubertal patients.\u003c/p\u003e \u003cp\u003eTherefore, the purpose of this study is to 1) understand the impact of the particularity of ovarian tissue structure of children on the fertility preservation by analyzing the morphological characteristics of ovarian tissue, 2) investigate the survival of ovarian tissues after vitrification and slow freezing, and 3) determine whether the inhibition of PTEN and activation of PI3K can activate follicles of children in vitro through an in vitro culture system.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy participants and ethical approval\u003c/h2\u003e \u003cp\u003e Use of human ovarian tissue was approved by Peking University Third Hospital Medical Science Research Ethics Committee (reference 2019SZ031). The study population included a total of 46 female children aged 1 to 14 years undergoing ovarian-sparing local mass excision from January 2019 to March 2025. The diagnosis of the patients included teratoma, ovarian cyst or other benign ovarian diseases. Ovarian tissue from 4 adult women was used as the controls. All patients or their guardians had provided written informed consent to donate their ovarian tissue to research.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSlow freezing and thawing\u003c/h3\u003e\n\u003cp\u003eThe slow freezing and thawing procedure was performed according to the method of Donnez et al (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). with a slight modification. Briefly, ovarian pieces were first equilibrated in freezing media, then individually transferred into cryovials containing 1.8 ml freezing media at 4\u0026deg;C. The cryovials were cooled in a programmable freezer (Kyro 360\u0026thinsp;\u0026minus;\u0026thinsp;1.7, Planer Inc., UK). For thawing, the cryovials were immersed in a water bath at 37\u0026deg;C with gentle shaking. When the liquid in the cryovials melted, tissues were transferred into thawing media.\u003c/p\u003e\n\u003ch3\u003eVitrification and thawing\u003c/h3\u003e\n\u003cp\u003eThe vitrification procedure was performed according to the method of Suzuki et al (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). with slight modifications. Ovarian pieces were vitrified using a two-step exposure to equilibration and vitrification solutions and then plunged into liquid nitrogen for storage. For thawing, samples were transferred to thawing media.\u003c/p\u003e\n\u003ch3\u003eOvarian tissue culture\u003c/h3\u003e\n\u003cp\u003eAfter thawing, ovarian tissues were cultured in 6-well plate by adding 4mL media. The culture media was as reported by wang(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) et al. All tissues were cultured at 37℃ under 5% CO\u003csub\u003e2\u003c/sub\u003e for up to 6 days. Every 2 days, half of the culture medium was replaced.\u003c/p\u003e\n\u003ch3\u003eIn vitro activation of primordial follicle\u003c/h3\u003e\n\u003cp\u003eOvarian tissues of IVA group were treated with 30\u0026micro;M bpv (MedChemExpress, HY-128693) and 150 \u0026micro;g/mL 740Y-P (MedChemExpress, HY-P0175) in culture media for 24h and only 150\u0026micro;g/mL 740Y-P for next 24h. For the control group, the culture media were replaced at the same time as the IVA group. Ovarian tissues were collected on day 2 of culture to evaluate the efficiency of follicular activation following IVA. To assess the subsequent developmental potential of activated follicles, tissues were harvested on day 6 of culture.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFollicle number Counting\u003c/h2\u003e \u003cp\u003eOvarian tissues were collected and fixed in 10% neutral buffered formalin fixative for 20 hours, then embedded in paraffin. Follicle quantification was performed following the protocol established by Yano Maher et al.(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Briefly, the paraffin blocks were cut into serial sections of 5 \u0026micro;m. Every 5th section was stained with hematoxylin and eosin (H.E.) for follicle classification and counting. Count the total number of follicles, and divide the number of follicles at each stage by the total number of follicles to obtain the distribution of follicles, expressed as a percentage. The primary follicle increasing rate is expressed as the difference between the proportion of primary follicles among total follicles after culture and that before culture, divided by the proportion before culture, and presented as a percentage. This parameter reflects the rate of increase in the proportion of primary follicles during the culture period, serving as an indicator of follicular activation efficiency.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetection and quantification of apoptosis\u003c/h3\u003e\n\u003cp\u003eFor TUNEL technology, tissue sections were dewaxed and rehydrated. Tissue sections were incubated for 1h with In Situ Cell Death Detection Kit, TMR Red (Roche, 12156792910, Germany), at 37℃ in a humidified atmosphere in the dark. Samples were analyzed by Opereta CLS (PerkinElmer, USA). In the same batch, prepare a positive control (by treating the sample with DNase I for 10 minutes at 37\u0026deg;C) and a negative control (by incubating only with the lable solution without adding TdT enzyme). The positive cell rate in the tissue is defined as the number of positive cells divided by the total number of cells, expressed as a percentage.\u003c/p\u003e\n\u003ch3\u003eImmunoblotting analysis\u003c/h3\u003e\n\u003cp\u003eOvarian proteins were extracted by Radio Immunoprecipitation Assay (RIPA) lysis buffer (ThermoFisher, 89901), containing Protease and Phosphatase Inhibitor Cocktail (ThermoFisher, 78442). A total of 20 \u0026micro;g proteins in each sample were loaded and separated by electrophoresis (Bio-Rad, USA, 1610732). After electronic transfer (Bio-Rad, 1610734), the Nitrocellulose Membrane (Millipore, Germany, HATF08130) were blocked in 5% blocking buffer (Bio-Rad, 12010020) for 10min and then incubated for 14h at 4\u0026deg;C with specific antibodies. Antibodies included p-FOXO3a (1:1000; Abcam, UK, ab47285), FOXO3a (1:2000, HUABIO, China, ER1706-79), and β-actin (1:2000, Cell Signaling Technology, USA, 4970). After washing with Tris Buffered Saline with Tween 20 (TBST) for three times, the Horseradish Peroxidase (HRP)-conjugated relative secondary antibodies were then used to detect proteins through enhanced chemiluminescence on the Tanon 5200 analysis system.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eAfter deparaffinization and rehydration, incubate the sections with 3% hydrogen peroxide diluted in methanol for 10 minutes at room temperature. Antigen retrieval was then carried out by heating the sections in 10 mM citrate buffer (pH 6.0). The tissue sections were subsequently incubated overnight at 4\u0026deg;C with primary antibodies p-FOXO3a (1:100). Sections were incubated with HRP-conjugated secondary antibodies and visualized using a DAB detection system. Follicles were classified based on the nuclear translocation of pFOXO3a: follicles showing nuclear localization of pFOXO3a were considered positive, while those without nuclear translocation were classified as negative.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using GraphPad Prism version 8.0 (GraphPad Software, USA). For all analyses, data were first assessed for normality and homogeneity of variance. For datasets that met the assumptions of normal distribution and equal variance, comparisons between two groups were conducted using the unpaired or paired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test, depending on whether the samples were independent or matched (derived from the same patient). Comparisons among three or more groups were performed using one-way analysis of variance (ANOVA). For non-normally distributed or heteroscedastic data, the Mann\u0026ndash;Whitney \u003cem\u003eU\u003c/em\u003e test was applied. Categorical (binary) outcomes were analyzed using contingency table (chi-square or Fisher\u0026rsquo;s exact) tests. A significance threshold of α\u0026thinsp;=\u0026thinsp;0.05 was adopted. Where applicable, 95% confidence intervals (CIs) and statistical power (effect size, post hoc power) were reported to support the robustness of the findings.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFollicular Composition and Abundance in Prepubertal Ovarian Tissue\u003c/h2\u003e \u003cp\u003eTo characterize the follicular composition of prepubertal ovarian tissue, we examined the distribution of follicle stages in freshly collected samples from forty-six girls. All the children had not menstruated. The patients\u0026rsquo; characteristics are reported in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The distribution of pediatric ovarian tissue samples across the experimental procedures is outlined in the flow diagram in Fig.\u0026nbsp;1. Antral follicles were observed in all age groups (Fig.\u0026nbsp;2B, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Primordial and primary follicles were the main types of ovarian follicles in children of all ages (Fig.\u0026nbsp;2A). The proportions of primordial follicles in the low (\u0026lt;\u0026thinsp;4 years old), medium (5\u0026ndash;8 years old), and high (\u0026gt;\u0026thinsp;9 years old) age groups of children's ovaries were 68.96% \u0026plusmn; 19.50%, 65.17% \u0026plusmn; 19.09%, and 54.13% \u0026plusmn; 20.50% (F\u0026thinsp;=\u0026thinsp;2.08, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.115, effect size η\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.12, 95%CI of η\u003csup\u003e2\u003c/sup\u003e: 0.04\u0026ndash;0.34). The proportions of primary follicles in the low, medium, and high age groups of children's ovaries were 26.73% \u0026plusmn; 13.31%, 36.34% \u0026plusmn; 25.10%, and 39.80% \u0026plusmn; 15.69% (F\u0026thinsp;=\u0026thinsp;1.88, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.15, effect size η\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.11, 95%CI of η\u003csup\u003e2\u003c/sup\u003e: 0.05\u0026ndash;0.31). These findings demonstrate that prepubertal ovarian tissue contains a rich follicular reserve dominated by primordial and primary follicles, suggesting that it represents an excellent substrate for activation and fertility preservation strategies.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 1. Flow diagram of sample allocation and experimental procedures.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis flow chart summarizes the allocation of ovarian tissue samples obtained from forty-six prepubertal girls undergoing ovarian-sparing surgery. All samples were first evaluated by fresh tissue morphology analysis. Following screening, tissues were assigned to cryopreservation by slow freezing or vitrification. Three ovarian tissue samples were too small to be divided into experimental fragments and therefore were not subjected to cryopreservation procedures. Five patients contributed matched samples for apoptosis assessment under fresh, slow-frozen, and vitrified conditions. Ten patients provided ovarian tissue for six-day in vitro culture to compare post-thaw developmental potential. Molecular activation analyses (pFOXO3a immunohistochemistry and Western blotting) were performed on thawed tissues derived from six donors involved in the activation experiments. In vitro activation experiments included nine patients in the slow freezing group and thirteen patients in the vitrification group.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 2. Fresh ovarian tissue histology in children of different ages.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e(A) Distribution of follicles in low-aged (0\u0026thinsp;~\u0026thinsp;3 years old, n\u0026thinsp;=\u0026thinsp;6), middle-aged (4\u0026thinsp;~\u0026thinsp;8 years old, n\u0026thinsp;=\u0026thinsp;18), and high-aged (over 9 years old, n\u0026thinsp;=\u0026thinsp;22) children ovaries. Data are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Pr, Primordial follicle; P, Primary follicle; S, Secondary follicle; A, Antral follicle. (B) Ovarian histology shown by hematoxylin and eosin staining of different ages and stages. Representative images of primordial follicles, primary follicles, secondary follicles, antral follicles were displayed. Except for those directly marked in the figure, all scale bars represent 50\u0026micro;m. The black arrows indicate the primordial follicles while the white arrows indicate the primary follicles.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e Fresh ovarian tissue histology in Adults.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOvarian histology shown by hematoxylin and eosin staining. Representative images of primordial follicles, primary follicles, secondary follicles, antral follicles were displayed. Except for those directly marked in the figure, all scale bars represent 50\u0026micro;m. The black arrows indicate the primordial follicles while the white arrows indicate the primary follicles.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePopulation characteristics\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003elow-age group\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003emiddle-age group\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;18\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ehigh-age group\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;22\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eage (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.8 (0.9 to 2.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.8 (4.8 to 7.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.0 (11.0 to 14.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eweight(kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.6 (10.0 to 14.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.5 (18.0 to 25.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e49.0 (33.0 to 62.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eheight(m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.79 (0.72 to 0.89)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.22 (1.18 to 1.29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.58 (1.47 to 1.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.57 (15.77\u0026ndash;18.76)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.30 (14.12 to 16.80)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.59 (16.84 to 24.88)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDiagnosis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMature teratoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOvarian cyst\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTeratoma comorbid with cyst\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eReproductive endocrine hormone\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFSH(IU/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.925 (0.100 to 2.430)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.955 (0.585 to 1.620)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.170 (2.150 to 6.190)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0198\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLH(IU/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.10 (0.10 to 0.18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.10 (0.10 to 0.13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.39 (0.33 to 3.55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0084\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE\u003csub\u003e2\u003c/sub\u003e(pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60.0 (42.3 to 199.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.5 ( 13.8 to 54.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50.0 (17.56 to 126.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.5976\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP(ng/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.64 (0.40 to 1.10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.68 (0.43 to 2.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.91 (0.43 to 1.30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.1932\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT(ng/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.69 (0.69 to 2.36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.69 (0.69 to 3.20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.88 (0.69 to 14.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0566\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePRL(ng/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.80 (7.31 to 50.88)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.15 (7.13 to 25.85)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.33 (10.14 to 37.48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.5620\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAMH(ng/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.71 (1.47 to 3.31)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.16 (1.31 to 5.12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.49 (2.88 to 6.01)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0573\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eData are shown as IQR [Median (Q1 to Q3)]. FSH: follicle-stimulating hormone; LH: luteinizing hormone; E2: estradiol; P: progesterone hormone; T: testosterone; PRL: prolactin; AMH: anti-mullerian hormone\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe distribution and morphology of different development stages of ovarian tissue after cryopreservation/thawing by different methods\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo compare the tissue viability following slow freezing and vitrification, apoptotic levels were analyzed in ovarian tissues from five patients under three conditions: fresh, slow freezing, and vitrification. The proportions of apoptotic cells in the three groups were 2.32%\u0026plusmn;2.70%, 13.91%\u0026plusmn;3.66%, and 49.91%\u0026plusmn;14.20%, showing a statistically significant difference (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0002). Compared with fresh ovarian tissue, both slow freezing and vitrification significantly increased apoptosis (Fresh vs. Sf: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0049, 95%CI: \u0026minus;16.3, \u0026minus;\u0026thinsp;6.8, Cohen\u0026rsquo;s \u003cem\u003ed\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.03; Fresh vs. Vit: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0054, 95%CI: \u0026minus;65.5-\u0026minus;29.7, Cohen\u0026rsquo;s \u003cem\u003ed\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.30). In addition, vitrification resulted in markedly higher apoptosis than slow freezing (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0042, 95%CI: \u0026minus;53.1-\u0026minus;18.9, Cohen\u0026rsquo;s \u003cem\u003ed\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.62). Meanwhile, staining results indicated that apoptotic cells were distributed in both follicles and stromal cells (Fig.\u0026nbsp;3A, E). To further assess follicular damage following different cryopreservation methods, the survival status of 311 follicles was analyzed using contingency table analysis. The proportions of follicular damage in the fresh, slow-freezing, and vitrification groups were 6.73%, 26.09%, and 59.65% (Fig.\u0026nbsp;3B, D). There is evidence indicating that slow freezing is associated with abnormal follicular morphology (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0004, 95%CI: 1.097\u0026ndash;1.565, relative risk RR\u0026thinsp;=\u0026thinsp;1.262). There is also evidence of an association between vitrification and follicular damage (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, 95% CI: 1.745\u0026ndash;3.265, relative risk RR\u0026thinsp;=\u0026thinsp;2.331). Additionally, the proportion of damaged follicles was significantly higher after vitrification compared to slow freezing (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0008, 95% CI: 1.294\u0026ndash;2.667, relative risk RR\u0026thinsp;=\u0026thinsp;1.832). The proportion of primordial follicles in ovarian tissue after thawing was 68.16% \u0026plusmn; 15.73% and 68.26% \u0026plusmn; 8.96% for slow freezing and vitrification methods, which was not significantly different from the fresh group (59.90% \u0026plusmn; 27.68%) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.3800, effect size η\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.079, 95%CI of η\u003csup\u003e2\u003c/sup\u003e: 0.022\u0026ndash;0.311,). The proportion of primary follicles in ovarian tissue after thawing was 31.12% \u0026plusmn; 15.11% and 30.29% \u0026plusmn; 6.91% for slow freezing and vitrification methods, which was not significantly different from the fresh group (38.09% \u0026plusmn; 26.57%) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.82, effect size η\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.05, 95%CI of η\u003csup\u003e2\u003c/sup\u003e: 0.006\u0026ndash;0.843). (Fig.\u0026nbsp;3C). These findings indicate that slow freezing causes less ovarian tissue damage compared to vitrification.\u003c/p\u003e \u003cp\u003eFurthermore, to evaluate the follicular developmental potential of ovarian tissue following slow freezing and vitrification, ovarian samples from ten patients were subjected to a 6-day in vitro culture. The increase rate of primary follicles in the slow freezing group was 14.41% \u0026plusmn; 7.51%, which was significantly higher than 5.13% \u0026plusmn; 5.16% in the vitrification group (U\u0026thinsp;=\u0026thinsp;22.0, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.038, 95%CI: 0.026\u0026ndash;0.164, Cohen\u0026rsquo;s \u003cem\u003ed\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.39) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These findings indicate that ovarian tissue subjected to slow freezing exhibits greater developmental potential during in vitro culture compared to vitrified tissue.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 3. Apoptotic and morphological assessment of prepubertal ovarian tissue following fresh processing, slow-freezing and vitrification.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e(A) Quantification of apoptotic cells by TUNEL assay in fresh (Fresh), slow-freezing (Sf) and vitrification (Vit) groups. Bars show mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM; Fresh vs. Sf, p\u0026thinsp;=\u0026thinsp;0.0049; Fresh vs. Vit, p\u0026thinsp;=\u0026thinsp;0.0054; Sf vs. Vit, p\u0026thinsp;=\u0026thinsp;0.0042. (B) Proportion of morphologically normal (black) and damaged (gray) follicles after each treatment. Cryopreservation significantly increased follicular damage (χ\u0026sup2; test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).(C) Distribution of follicle developmental stages\u0026mdash; primordial (Pr), primary (P), secondary (S) and antral (A)\u0026mdash;in each group. No marked shifts in stage composition were observed following either slow‐freezing or vitrification.(D) Representative hematoxylin \u0026amp; eosin\u0026ndash;stained sections of ovarian cortex after processing under each condition. Scale bar =\u0026thinsp;50 \u0026micro;m.(E) Representative fluorescence images of TUNEL assay showing nuclei (DAPI, blue) and apoptotic cells (TUNEL, yellow). Scale bar =\u0026thinsp;100 \u0026micro;m.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eIVA enhances follicular activation and early development in thawed prepubertal ovarian tissue\u003c/h2\u003e \u003cp\u003eUsing the PTEN inhibitor bpv and PI3K activator 740Y-P, IVA can activate primordial follicles from frozen-thawed pediatric ovarian tissues, offering a promising strategy for preserving fertility in children. Due to the presence of spontaneously activated first-wave follicles in pediatric ovarian tissue, it remains unclear whether the adult IVA protocol is capable of activating primordial follicles in pediatric ovarian tissue. To address this question, pediatric ovarian tissues subjected to different cryopreservation methods were treated with IVA under a defined culture system.\u003c/p\u003e \u003cp\u003eTo confirm molecular changes associated with activation, Western blot analysis was performed to quantify the ratio of phosphorylated FOXO3a to total FOXO3a (pF/F). The slow freezing IVA group showed a significantly higher pF/F compared to the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0078, mean difference: 0.68, 95%CI: -1.04 to -0.32, Cohen\u0026rsquo;s \u003cem\u003ed\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.65), as well as the vitrification group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002, mean difference: 1.75, 95%CI: -2.24 to -1.25, Cohen\u0026rsquo;s \u003cem\u003ed\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.73) (Fig.\u0026nbsp;4C-3F). These findings suggest that the activation protocol using bpV/740Y-P is effective in activating the primordial follicles of cryopreserved pediatric ovarian tissue.\u003c/p\u003e \u003cp\u003eTo evaluate follicular activation following IVA, ovarian tissues were first analyzed for pFOXO3a expression and localization by day 2 of culture (Fig.\u0026nbsp;4A-3B). In the slow freezing group, a total of 142 follicles were analyzed. Following IVA, 64.89% of follicles exhibited nuclear translocation of pFOXO3a, compared to 25.00% in the control group, indicating that there is evidence of an association between IVA and pFOXO3a nuclear translocation (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, 95%CI: 0.2257\u0026ndash;0.6162, relative risk RR\u0026thinsp;=\u0026thinsp;2.596). Similarly, in the vitrification group, 279 follicles were included, with 79.25% of follicles showing nuclear translocation after IVA, compared to 39.58% in the control group, suggesting that IVA significantly enhances pFOXO3a nuclear translocation during post-vitrification culture (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, 95%CI: 0.3975\u0026ndash;0.6166, relative risk RR\u0026thinsp;=\u0026thinsp;2.002). Furthermore, contingency table analysis was performed to compare the proportion of follicles exhibiting nuclear translocation after IVA between slow-frozen and vitrified ovarian tissues. The results demonstrated that the incidence of nuclear translocation was significantly higher following slow freezing compared to vitrification (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.022, 95%CI: 1.038\u0026ndash;1.470, relative risk RR\u0026thinsp;=\u0026thinsp;1.221).\u003c/p\u003e \u003cp\u003eTo further investigate the follicular development potential of cryopreserved ovarian tissue following IVA, ovarian culture outcomes were analyzed from nine patients in the slow-freezing group and thirteen patients in the vitrification group. Representative morphological features of the cultured tissues are shown in Fig.\u0026nbsp;4G, H. In slow freezing group, IVA significantly increased primary follicle increasing rate compared to control group (47.09%\u0026plusmn;19.36% vs. 22.60%\u0026plusmn;5.97%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.031, 95%CI: 0.10\u0026ndash;1.52, Cohen\u0026rsquo;s \u003cem\u003ed\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.13). Similarly, in vitrification group, IVA significantly increased primary follicle increasing compared to control group (65.93%\u0026plusmn;64.25% vs. 23.58%\u0026plusmn;67.26%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005, 95%CI: 0.06\u0026ndash;0.82, Cohen\u0026rsquo;s \u003cem\u003ed\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.71). Further analysis of the proportions of follicles at each developmental stage is shown in Fig.\u0026nbsp;4I, 4J. In the slow freezing group, the proportion of primordial follicles was significantly reduced in the activation group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0005, 95%CI: 0.1072\u0026ndash;0.3930), while the proportion of primary follicles was significantly higher in the activation group compared to the control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0012, 95%CI: -0.3713- -0.0855). In contrast, there is no statistically significant differences were observed between the activation and control groups in terms of follicular stage distribution in vitrification group. These results indicate that IVA promotes follicular development during culture, but there is no evidence of difference between the two cryopreservation methods.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 4. IVA promotes the activation of primordial follicles in children's ovaries after cryopreservation-thawing.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e(A) H.E. staining of ovarian cortical sections from vitrified (Vit) and slow-frozen (Sf) tissues, either without activation (NC) or following IVA. (B) Immunohistochemical detection of phosphorylated FOXO3a (pFOXO3a) in the same four groups. Scale bars, 50 \u0026micro;m. (C, D) Quantification of pFOXO3a‐positive (black) versus pFOXO3a‐negative (gray) follicles in Vit and Sf groups. IVA markedly increased the proportion of pFOXO3a‐positive follicles in both Vit and Sf tissues (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, χ\u0026sup2; test). (E, F) Representative Western blots for pFOXO3a, total FOXO3a and β‐actin in NC versus IVA samples of Vit and Sf tissues. Densitometric analysis of the pFOXO3a/FOXO3a ratio, showing significant elevation of FOXO3a phosphorylation after IVA in both Vit (p\u0026thinsp;=\u0026thinsp;0.0140) and Sf (p\u0026thinsp;=\u0026thinsp;0.0357) groups (Student\u0026rsquo;s t‐test). (G) H.E. stained sections of vitrified (Vit) ovarian cortex in fresh (immediately fixed), non-activated control (NC) and IVA groups. Right: percentage increase in primary follicles following IVA (p\u0026thinsp;=\u0026thinsp;0.0286). (H) H.E. stained for slow-frozen (Sf), showing a similar IVA-\u003c/p\u003e \u003cp\u003einduced rise in primary follicles (p\u0026thinsp;=\u0026thinsp;0.0331). Scale bars, 50 \u0026micro;m. (I) Corresponding follicle distribution for Vit tissues, revealing no significant difference in stage proportions after IVA. (J) Follicle-stage distribution in Sf tissues: bar graph of mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD percentages of primordial, primary and secondary follicles in NC (black) versus IVA (gray) groups; IVA significantly reduced primordial (p\u0026thinsp;=\u0026thinsp;0.0005) and increased primary (p\u0026thinsp;=\u0026thinsp;0.0012) follicle proportions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRepresentative Western blot membranes for the vitrification (Vit) and slow-freezing (Sf) groups. Each membrane shows protein expression in negative control (NC) and in vitro activation (IVA) samples (N\u0026thinsp;=\u0026thinsp;3). For each group, the original membrane was cut horizontally at the midline to allow incubation with different primary antibodies.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we described the distribution of follicles at all stages in fresh ovarian tissue from children, investigated the effectiveness of slow freezing and vitrification on child ovarian tissues, and examined the applicability of PTEN/PI3K IVA to primordial follicles of pediatric ovarian tissues. The results indicate that follicles at various developmental stages are present in the cortex of pediatric ovarian tissue. Slow freezing causes less damage to the ovarian tissue compared to vitrification. Moreover, the current adult IVA protocol is capable of activating primordial follicles in prepubertal ovarian tissue.\u003c/p\u003e \u003cp\u003eBased on available reports, this study appears to be among the more extensive analyses of fresh ovarian tissue morphology in prepubertal patients. Previous studies on the proportion of follicles at various stages in ovaries of children have been hindered due to the difficulty in obtaining samples (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), and the medulla of the underaged ovarian tissue contains a large number of activated first-wave follicles (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). In addition, we found that there were antral follicles in the ovaries of 1-year-old children. This provided new insights into the developmental potential of the first wave of follicles, suggesting that the follicles of early children have the potency to develop into antral follicles. Although the fertility of the anthropic first wave of follicles has been controversial, studies in mice suggest that these follicles have the ability to produce embryos through in vitro fertilization and could be targeted for human fertility preservation (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Overall, the abundance of follicles in prepubertal ovarian tissue indicates a favorable baseline for activation and offers a compelling rationale for prioritizing tailored preservation strategies in young patients.\u003c/p\u003e \u003cp\u003eThe efficacy of slow freezing and vitrification remains contentious. In this study, we reported a lower level of damaged follicles of slow frozen ovarian tissue of children than that of the vitrificated group after thawing, which was consistent with the findings of Dolmans et al. based on adult ovarian tissue(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), indicating the advantageous preservative capacity of slow freezing in follicle viability. Recently, the vital role of the stromal cell in follicle growth and development has been gradually recognized (\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Our study found that both vitrification and slow freezing led to increased levels of apoptosis of stromal cells compared to fresh tissue, with vitrification inducing a higher degree of apoptosis than slow freezing. Prolonged in vitro culture may contribute to reduced follicular developmental competence(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). We speculate that damage to stromal cells may be associated with the reduced in vitro developmental potential of follicles in vitrified-thawed ovarian tissue. Some studies have discussed vitrification as an alternative to slow freezing (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). However, evidence derived from adults should be cautiously extrapolated to children, especially considering that pediatric ovarian tissue preservation usually requires longer storage periods than adults. We recommend that capable centers should offer both cryopreservation methods concurrently for pediatric patients, taking into account the best interests of them.\u003c/p\u003e \u003cp\u003eIVA can activate primordial follicles. However, current study has not explored whether IVA can alter the fate of \"the first wave\" follicles destined for apoptotic atresia (\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Nuclear translocation of pFOXO3a, the increased pF/F ratio, and the increased proportion of primary follicles during in vitro culture collectively support the efficacy of IVA in pediatric ovarian tissue. These findings suggest that despite the presence of the naturally occurring \"first-wave\" follicle activation in pediatric ovaries, (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e), the follicles remain responsive to IVA, indicating that this approach may have applicability and practical value in pediatric ovarian tissue. Our study did not further investigate individual differences in follicular response to IVA across various age groups. Future studies could explore stratified activation strategies.\u003c/p\u003e \u003cp\u003eIn this study, follicle density was not reported because the ovarian tissues were obtained adjacent to benign lesions, and the amount of remaining healthy cortex varied considerably among patients. In several samples, the follicle density was extremely low due to limited residual tissue rather than true biological differences. Including these values would have introduced substantial variability and made the data difficult to interpret. Instead, we focused on the proportion of follicles at each developmental stage, which provides a more reliable indicator of follicular activation and developmental shifts in this cohort. Meanwhile, this study was limited to in vitro culture experiments, and some studies(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) pointed out that the transplantation process is another essential factor limiting the live birth rate of OTC, so further in vivo studies are needed to explore the optimal scheme of child fertility preservation.\u003c/p\u003e \u003cp\u003eIn summary, the abundant early follicles and clear activation potential of prepubertal ovarian tissue highlight the opportunity\u0026mdash;and necessity\u0026mdash;to refine cryopreservation and activation strategies specifically for pediatric fertility preservation. IVA serves as an important complementary strategy for fertility preservation in pediatric patients and should be considered in clinical practice. The significance of this study lies in providing new technological strategies for fertility preservation in pediatric patients. By combining multiple techniques, it is possible to achieve optimal protection and promotion of fertility in children.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eUse of human ovarian tissue was approved by Peking University Third Hospital Medical Science Research Ethics Committee (reference 2019SZ031). All study procedures adhered to the ethical principles outlined in the \u003cem\u003eDeclaration of Helsinki\u0026nbsp;\u003c/em\u003eand complied with the \u003cem\u003eAdministrative Measures for Ethical Review of Life Science and Medical Research Involving Humans\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eWritten informed consent for participation in the study and publication of anonymized data was obtained from all adult participants and from the parents or legal guardians of all pediatric participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eNational Natural Science Foundation of China (T2293764), National Key R\u0026amp;D Program of China (2022YFC2703000, 2022YFC2704400, 2022YFC2704401), and National Natural Science Foundation of China (82288102, 82171630).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.Y., Z.Z., M.Z. and M.T. contributed equally as first authors and HY.W., H.W. and J.Y. contributed equally as co-corresponding authors to the paper. All authors contributed to manuscript editing and read and approved the final manuscript. J.Y., Z.Z. and J.Y. conceived the project; HY.W., H.W., J.Q. and J.Y. supervised the study; M.Z. and J.Y. were responsible for the data curation. M.Z., M.T., J.H., T.L., J.H. and J.Y. performed the experiments; Z.Z., W.Y., H.C., S.Y., X.S., Y.W. and H.W performed the operations. J.Y., M.Z., M.T. and J.H. analyzed most experiments and compiled the figures. J.Y. and Z.Z. wrote the manuscript with edits from J.Y. and J.Q. HY.W, J.Q., J.Y., and Y.W. contributed to obtaining funding.\u003c/p\u003e\n\u003cp\u003eAcknowledgement\u003c/p\u003e\n\u003cp\u003eWe want to thank Dr. Zheng Wang for reviewing the English language of the article. We are grateful to Dr. Yan Liu (Peking University Health Science Center) for her guidance on follicular morphology. We are grateful to Jinghao Yan, Yu Lin, Saishuo Chang, Fuyi Zhu and the other colleagues in the operating theater (National Center for Children\u0026apos;s Health) for their help in preserving and transporting clinical samples. We also express our appreciation to Jia Yang and Fengrui Yang (Peking University Health Science Center) for their work on follicular counting.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSmith MA, Altekruse SF, Adamson PC, Reaman GH, Seibel NL. Declining Childhood and Adolescent Cancer Mortality. Cancer. 2014;120(16):2497-506.\u003c/li\u003e\n \u003cli\u003eKlipstein S, Fallat ME, Savelli S, Bioethics C, Oncology SH, Surg S. Fertility Preservation for Pediatric and Adolescent Patients With Cancer: Medical and Ethical Considerations. Pediatrics. 2020;145(3):e20193994.\u003c/li\u003e\n \u003cli\u003eLevine JM, Whitton JA, Ginsberg JP, Green DM, Leisenring WM, Stovall M, et al. 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Oocyte-specific deletion of causes premature activation of the primordial follicle pool. Science. 2008;319(5863):611-3.\u003c/li\u003e\n \u003cli\u003eLi J, Kawamura K, Cheng Y, Liu S, Klein C, Liu S, et al. Activation of dormant ovarian follicles to generate mature eggs. Proc Natl Acad Sci U S A. 2010;107(22):10280-4.\u003c/li\u003e\n \u003cli\u003eHao J, Li T, Heinzelmann M, Moussaud-Lamodi\u0026egrave;re E, Lebre F, Krjut\u0026scaron;kov K, et al. Effects of chemical in vitro activation versus fragmentation on human ovarian tissue and follicle growth in culture. Human Reproduction Open. 2024;2024(3).\u003c/li\u003e\n \u003cli\u003eZhang L, Zhang J, Zhai J, Liu XC, Deng WF, Wang H, et al. Autotransplantation of the ovarian cortex after activation for infertility treatment: a shortened procedure. Human Reproduction. 2021;36(8):2134-47.\u003c/li\u003e\n \u003cli\u003eSun N, Li Z, Pang W, Wang L, Li W. Live birth after transplantation of cryopreserved ovarian tissue with two-year follow-tup: report of the first Chinese case. Chinese Journal of Reproduction and Contraception. 2021;41(11):1026-30.\u003c/li\u003e\n \u003cli\u003eDonnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, et al. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. The Lancet. 2004;364(9443):1405-10.\u003c/li\u003e\n \u003cli\u003eSuzuki N, Yoshioka N, Takae S, Sugishita Y, Tamura M, Hashimoto S, et al. Successful fertility preservation following ovarian tissue vitrification in patients with primary ovarian insufficiency. Human Reproduction. 2015;30(3):608-15.\u003c/li\u003e\n \u003cli\u003eWang T-r, Yan J, Lu C-l, Xia X, Yin T-l, Zhi X, et al. Human single follicle growth in vitro from cryopreserved ovarian tissue after slow freezing or vitrification. Human Reproduction. 2016;31(4):763-73.\u003c/li\u003e\n \u003cli\u003eYano Maher JC, Zelinski MB, Oktay KH, Duncan FE, Segars JH, Lujan ME, et al. Classification system of human ovarian follicle morphology: recommendations of the National Institute of Child Health and Human Development - sponsored ovarian nomenclature workshop. Fertility and Sterility. 2025;123(5):761-78.\u003c/li\u003e\n \u003cli\u003eAnderson RA, McLaughlin M, Wallace WHB, Albertini DF, Telfer EE. The immature human ovary shows loss of abnormal follicles and increasing follicle developmental competence through childhood and adolescence. Human Reproduction. 2013;29(1):97-106.\u003c/li\u003e\n \u003cli\u003eForabosco A, Sforza C. Establishment of ovarian reserve: a quantitative morphometric study of the developing human ovary. Fertility and Sterility. 2007;88(3):675-83.\u003c/li\u003e\n \u003cli\u003eSforza C, Vizzotto L, Ferrario VF, Forabosco A. Position of follicles in normal human ovary during definitive histogenesis. Early Human Development. 2003;74(1):27-35.\u003c/li\u003e\n \u003cli\u003eMan LM, Guahmich NL, Kallinos E, Park L, Bodine R, Zaninovic N, et al. Xenograft model of heterotopic transplantation of human ovarian cortical tissue and its clinical relevance. Reproduction. 2023;165(1):31-47.\u003c/li\u003e\n \u003cli\u003eKristensen SG, Rasmussen A, Byskov AG, Andersen CY. Isolation of pre-antral follicles from human ovarian medulla tissue. Human Reproduction. 2011;26(1):157-66.\u003c/li\u003e\n \u003cli\u003eDalman A, Farahani NSDG, Totonchi M, Pirjani R, Ebrahimi B, Valojerdi MR. Slow freezing versus vitrification technique for human ovarian tissue cryopreservation: An evaluation of histological changes, WNT signaling pathway and apoptotic genes expression L. Cryobiology. 2017;79:29-36.\u003c/li\u003e\n \u003cli\u003eAyuandari S, Winkler-Crepaz K, Paulitsch M, Wagner C, Zavadil C, Manzl C, et al. Follicular growth after xenotransplantation of cryopreserved/thawed human ovarian tissue in SCID mice: dynamics and molecular aspects. Journal of Assisted Reproduction and Genetics. 2016;33(12):1585-93.\u003c/li\u003e\n \u003cli\u003eHopkins TIR, Bemmer VL, Franks S, Dunlop C, Hardy K, Dunlop IE. Micromechanical mapping of the intact ovary interior reveals contrasting mechanical roles for follicles and stroma. Biomaterials. 2021;277:121099.\u003c/li\u003e\n \u003cli\u003eKinnear HM, Tomaszewski CE, Chang FL, Moravek MB, Xu M, Padmanabhan V, et al. The ovarian stroma as a new frontier. Reproduction. 2020;160(3):R25-R39.\u003c/li\u003e\n \u003cli\u003eGuo YC, Xue LR, Tang WC, Xiong JQ, Chen D, Dai Y, et al. Ovarian microenvironment: challenges and opportunities in protecting against chemotherapy-associated ovarian damage. Human Reproduction Update. 2024;30(5):614-47.\u003c/li\u003e\n \u003cli\u003eHossay C, Tramacere F, Camboni A, Cacciottola L, Van Kerk O, Donnez J, et al. P-437\u0026emsp;Follicle activation in human ovarian tissue: impact of freezing, culture and grafting. Human Reproduction. 2022;37(Supplement_1):deac107.412.\u003c/li\u003e\n \u003cli\u003eShi QQ, Xie YD, Wang Y, Li SW. Vitrification versus slow freezing for human ovarian tissue cryopreservation: a systematic review and meta-anlaysis. Scientific Reports. 2017;7(1):8538.\u003c/li\u003e\n \u003cli\u003eHirshfield AN. Heterogeneity of Cell Populations that Contribute to the Formation of Primordial Follicles in Rats1. Biology of Reproduction. 1992;47(3):466-72.\u003c/li\u003e\n \u003cli\u003eErnst EH, Franks S, Hardy K, Villesen P, Lykke-Hartmann K. Granulosa cells from human primordial and primary follicles show differential global gene expression profiles. Human Reproduction. 2018;33(4):666-79.\u003c/li\u003e\n \u003cli\u003eRooda I, Hassan J, Hao J, Wagner M, Moussaud-Lamodi\u0026egrave;re E, J\u0026auml;\u0026auml;ger K, et al. In-depth analysis of transcriptomes in ovarian cortical follicles from children and adults reveals interfollicular heterogeneity. Nature Communications. 2024;15(1):6989.\u003c/li\u003e\n \u003cli\u003eAlbamonte MI, Calabro LY, Albamonte MS, Vitullo AD. FOXO3 and PTEN expression in the ovary of girls with extra-gonadal cancer with or without chemotherapy treatment prior to cryopreservation. Bmc Womens Health. 2023;23(1):509.\u003c/li\u003e\n \u003cli\u003eTarnawa ED, Baker MD, Aloisio GM, Carr BR, Castrillon DH. Gonadal expression of Foxo1, but not Foxo3, is conserved in diverse Mammalian species. Biol Reprod. 2013;88(4):103.\u003c/li\u003e\n \u003cli\u003eDamous LL, Nakamuta JS, Soares-Jr JM, Maciel GAR, Sim\u0026otilde;es RdS, Montero EFdS, et al. Females transplanted with ovaries subjected to hypoxic preconditioning show impair of ovarian function. Journal of Ovarian Research. 2014;7(1):34.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pediatric ovarian tissue, cryopreservation, slow freezing, vitrification, in vitro activation, fertility preservation","lastPublishedDoi":"10.21203/rs.3.rs-8321884/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8321884/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e \u003cp\u003eFertility preservation in pediatric patients requires effective cryopreservation of ovarian tissue, yet the relative performance of slow freezing and vitrification in this age group remains unclear. This study compared the two cryopreservation methods in prepubertal ovarian tissue and examined their functional consequences through in vitro activation and short-term culture.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e \u003cp\u003eOvarian tissues from forty-six girls aged one to fourteen years were processed fresh, slow-frozen, or vitrified. Follicular morphology and developmental stage assessed by hematoxylin and eosin staining; apoptosis assessed by TUNEL assay; follicular activation assessed by Western blotting and immunohistochemistry; follicular growth after IVA culture. Appropriate statistical analyses were applied based on data distribution, including t-tests, non-parametric tests, and contingency table analyses.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e \u003cp\u003eSlow freezing preserved follicular morphology better than vitrification, with significantly less oocyte damage and stromal cell apoptosis. IVA significantly increased pFOXO3a/FOXO3a ratios at day 2 in both groups and promoted nuclear translocation of pFOXO3a. By day 6, IVA-treated tissues showed a higher increase in primary follicle numbers compared to untreated controls, particularly in the slow freezing group.\u003c/p\u003e\u003ch2\u003eConclusions:\u003c/h2\u003e \u003cp\u003eSlow freezing is more effective than vitrification for pediatric ovarian tissue cryopreservation. IVA further enhances follicular activation after thawing and may provide a promising strategy for optimizing pediatric fertility preservation.\u003c/p\u003e","manuscriptTitle":"Improving Follicle Development After Cryopreservation of Prepubertal Ovarian Tissue Through In Vitro Activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-19 11:37:27","doi":"10.21203/rs.3.rs-8321884/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f9e36479-767d-46bc-a81c-b7276c05670d","owner":[],"postedDate":"February 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-27T21:09:46+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-19 11:37:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8321884","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8321884","identity":"rs-8321884","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}