Abstract Background Mesenchymal stem cells (MSCs) have shown potential in repairing chemotherapy-induced premature ovarian failure (POF). However, challenges such as stem cell loss and immune phagocytosis post-transplantation hinder their application. Due to easy and safe handling, in vitro ovarian culture is widely available for drug screening, pathophysiological research, and in vitro fertilization. MSCs could exhibit therapeutic capacity for ovarian injury, and avoid stem cell loss and immune phagocytosis in vitro tissue culture system. Therefore, this study utilizes an in vitro ovarian culture system to investigate the reparative potential of human umbilical cord mesenchymal stem cells (hUCMSCs) and their mechanism. Methods In this study, a chemotherapy-induced POF model was established by introducing cisplatin in vitro ovarian culture system. The reparative effects of hUCMSCs on damaged ovarian tissue were validated through Transwell chambers. Tissue histology examination, immunohistochemical staining, Western blotting, and RT-PCR were employed to evaluate the expression effects of hUCMSCs on ferroptosis and fibrosis-related genes during the process of repairing cisplatin-induced POF. Results Cisplatin was found to activate ovarian follicles in vitro POF model. Transcriptomic sequencing analysis revealed that cisplatin could activate genes associated with ferroptosis. hUCMSCs alleviated cisplatin-induced POF by suppressing the expression of ferroptosis. Moreover, inhibiting ferroptosis by hUCMSCs also ameliorated ovarian hormone levels and reduced the expression of fibrosis-related factors α-SMA and COL-I in the ovaries. Conclusions This study confirms that cisplatin-induced ovarian damage via ferroptosis in vitro POF model, and hUCMSCs repair ovarian injury by inhibiting the ferroptosis pathway and suppressing fibrosis. This research contributes to evaluating the effectiveness of hUCMSCs in treating chemotherapy-induced POF by inhibiting ferroptosis in an in vitro ovarian culture system and provides a potential therapeutic strategy for chemotherapy-induced POF. Supplementary Information The online version contains supplementary material available at 10.1186/s12958-024-01310-x. Keywords: Umbilical cord mesenchymal stem cells, Premature ovarian failure, In vitro ovarian culture system, Ferroptosis, Fibrosis Introduction Premature ovarian failure (POF) is a prevalent gynecological disease and the primary cause of infertility in women below the age of 40. The pathogenesis of POF is intricate and involves multiple factors. Chemotherapeutic agents, particularly cisplatin, have been shown to reduce ovarian follicular reserve and contribute to the development of POF [[38]1, [39]2]. Cisplatin induces a decline in ovarian follicular reserve in cancer patients, characterized by rapid depletion of the primordial follicle pool [[40]3]. The ovarian damage instigated by cisplatin includes both structural and hormonal disruptions, resulting in menstrual irregularities, amenorrhea, infertility, and associated cognitive impairments, profoundly affecting life quality [[41]4]. Therefore, addressing the POF induced by chemotherapy remains an urgent issue. Mesenchymal stem cells (MSCs) are a type of multipotent stem cell widely used in repairing damaged tissue due to their ease of isolation from tissues, as well as their self-renewal and differentiation capabilities. MSCs transplantation is emerging as a novel therapeutic approach for tissue injuries [[42]5]. Several studies conducted in animal models of ovarian damage suggest that administering MSCs obtained from different cell types effectively improves ovarian function and restores POF [[43]6–[44]8]. Umbilical cord mesenchymal stem cells (UCMSCs), have been demonstrated to exhibit typical features of mesenchymal stem cells, and they ameliorate ovarian tissue damage by upregulating follicle-stimulating hormone receptor levels [[45]9, [46]10]. Additionally, UCMSCs increase ovarian weight, plasma E2 levels, and the number of standard follicles, effectively restoring ovarian function [[47]11]. Research findings have demonstrated that UCMSCs hold potential for repairing chemotherapy-induced POF [[48]12], mitigating the adverse effects of chemotherapy drugs, and improving fertility in POF patients [[49]13, [50]14]. Ferroptosis is a novel form of cell death characterized by iron (Fe)-dependent accumulation of reactive oxygen species (ROS), leading to lipid peroxidation-related cell demise [[51]15]. The mechanism underlying chemotherapy-induced ovarian dysfunction has been elucidated, highlighting the significant role of granulosa cell (GC) ferroptosis in POF [[52]16, [53]17]. During follicle development, GCs undergo rapid proliferation, and chemotherapy drugs may induce follicular atresia by directly inhibiting GC proliferation, prematurely activating primordial follicles, and depleting the follicular reserve [[54]18]. Recent studies indicate that cisplatin impairs ovarian function through excessive ROS-induced ferroptosis, while simultaneously affecting GC ferroptosis, resulting in impaired follicular development and ovarian tissue fibrosis, which may contribute to ovarian damage [[55]19–[56]21]. There exists a close relationship between ferroptosis and fibrosis, where cellular ferroptosis exacerbates tissue fibrosis, leading to further tissue damage, while inhibiting ferroptosis might aid in alleviating the fibrotic process [[57]22]. Recent research has demonstrated that MSCs could protect against ferroptosis, and enhance their therapeutic effectiveness [[58]23, [59]24]. Additionally, 3D human umbilical cord MSC spheroids have shown superior resistance to autophagy and apoptosis of granulosa cells in a POF rat model [[60]25]. MSC-derived exosomes restore cyclophosphamide-induced ovarian damage and granulosa cell apoptosis by inhibiting ferroptosis in a POF mouse model [[61]26]. Although many studies explore stem cell treatment in POF and ferroptosis, few focus on the mechanism in vitro POF model. In vitro tissue culture systems are regarded as promising and physiologically closer systems, offering superior simulation capabilities and a more accurate representation of the in vivo growth environment compared to traditional two-dimensional culture systems [[62]27, [63]28]. In vitro tissue culture systems are widely available for drug screening, pathophysiological research, personalized therapy, and regenerative medicine. In vitro ovarian culture systems, the primordial follicles retain the interactions between the follicles and also between the follicle and surrounding stroma, therefore follicular development initiates faster [[64]29]. Due to its easy and safe handling, the in vitro ovarian culture systems were employed to induce follicle growth and reconstitute the ovarian microenvironment [[65]30, [66]31]. In this study, we conducted the in vitro ovarian culture systems to assess the cisplatin-induced POF, and utilized hUCMSCs for repairing the damaged ovaries. Further, we explore the mechanism of hUCMSCs to restore chemotherapy-induced POF through ferroptosis. Materials and methods MSC cultivation and isolation hUCMSCs were isolated from human umbilical cord tissues as previous study [[67]32]. Briefly, hUCMSCs were isolated from human umbilical cord tissues of healthy parturient. hUCMSCs were cultured at 37 °C in an atmosphere with 5% CO[2] using DMEM/F12 (Gibco, USA) medium supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 100 IU/ml penicillin, and 100 µg/ml streptomycin. The culture medium was changed every 2 days until hUCMSCs reached the fifth passage. Animals Eight-week-old female ICR mice were purchased from the Laboratory Animal Center of Anhui Medical University and were subjected to a 3-day acclimation period under temperature (24 °C ± 2 °C), humidity (55% ± 5%), 12-hour light/dark, and sterile water conditions with free access to water and food before the studies were conducted. At the end of the acclimation period, the utilization of carbon dioxide anesthesia enables the implementation of the cervical dislocation method to be performed on individuals under deep anesthesia. All animal experiments were approved by the Institutional Animal Care and Use Committee of Anhui Medical University. In vitro POF model and hUCMSCs treatment The 8-week-old female ICR mice were sacrificed to collect the ovaries. DMEM/F-12 medium and Matrigel (BD Bio-coat, Corning, USA) were mixed in a 1:1 volume ratio and used to support ovarian culture in vitro, with the ovary embedded in the mixed matrix gel (Fig. [68]1). The phase transition of the matrix gel from sol to gel occurred at 37℃ for 30 min. Cisplatin was then added to the culture medium at a concentration of 30 µM. The in vitro POF model was established after treatment with cisplatin for 48 h. The hUCMSCs were seeded in 24-well plates (3 × 10^5 cells/mL), and when the cell density was 60-70%, the ovaries wrapped in mixed matrix gel were transferred into the plates. Transwell chambers were used to separate the two; the permeable polycarbonate membrane allowed substances secreted by hUCMSCs to affect the ovarian tissue in the upper chamber without the entire ovary migrating to the lower chamber. Ovaries were randomly divided into 5 groups: Control group, Cisplatin group, Erastin group, MSC group, and Fer-1 group. The ovaries were categorized into six groups for vitro culture: (i) Control group (CTR), ovaries enveloped in the matrix gel; (ii) Cisplatin group (CIS), ovaries treated with cisplatin in the presence of the matrix gel; (iii) Erastin group (Erastin) ovaries treated with the Erastin (ferroptosis inducer) in the presence of the matrix gel; (iv) MSC group (CIS + MSC), ovarian tissue repair using hUCMSCs after cisplatin treatment; (v) Fer-1 group (CIS + Fer-1), ovarian tissue repair using the Fer-1 (ferroptosis inhibitor) after cisplatin treatment. Fig. 1. [69]Fig. 1 [70]Open in a new tab Schematic description of the experimental design. The ovaries of 8-week-old female rats were encapsulated in Matrigel and placed in a Transwell chamber. An in vitro POF model was established by exposing the ovaries to cisplatin for 2 days. Afterward, hUCMSCs were used for repair ([71]www.figdraw.com) Hematoxylin and Eosin (H&E) staining and follicle counting After 4 days of in vitro ovarian culture, the ovaries were collected for histological observation. They were fixed in 4% formaldehyde for 24 h and embedded in paraffin. Sections of 5 μm thickness were prepared from paraffin and stained with Saffron-Hematoxylin and Eosin (H&E) to observe follicular growth. All follicles within the entire ovary were counted at a 200x magnification field. The classification of follicles in the ovarian tissue included: Primordial follicles (primary follicles), typically situated in the superficial layer of the ovarian cortex, consisting of oocyte cells enveloped by a monolayer of flat granulosa cells; Primary follicles, larger than primordial follicles, composed of oocyte cells surrounded by a monolayer of cuboidal granulosa cells; Secondary follicles, comprising oocyte cells encircled by two or more layers of cuboidal granulosa cells; Antral follicles, characterized by a cavity filled with follicular fluid; Corpus luteum, consisting of luteal cells. Masson staining 4 μm paraffin-embedded murine ovary tissue sections were sequentially dewaxed in 3 xylenes, rehydrated in ethanol, and rinsed with distilled water, following the manufacturer’s instructions (Solarbio, China), paraffin sections were subjected to Masson’s trichrome staining. The evaluation of ovarian fibrosis levels was conducted by calculating the percentage of blue-stained areas (representing collagen) within the tissue. Immunohistochemistry After deparaffinization and rehydration of ovarian paraffin sections in xylene and ethanol gradients, immunohistochemical staining was conducted. According to the instructions provided in the immunohistochemistry kit (Solarbio, China), the sections were initially subjected to heat-induced antigen retrieval using a sodium citrate solution, followed by blocking with H[2]O[2] and 10% goat serum to block all sections. Subsequently, the sections were incubated overnight with the primary antibody at 4 °C, followed by incubation with the secondary antibody for 1 h at room temperature. 3,3’-Diaminobenzidine (DAB) was used as the chromogen and incubated for 1 min at room temperature, followed by counterstaining with hematoxylin for 10 s to stain the cell nuclei. Between each step, the samples were washed three times with PBS to ensure thorough cleaning and accurate staining. Positive staining regions typically appeared brown. Each ovary was observed under a microscope at 200x magnification, and the positive staining areas were quantified using ImageJ software. The final results were calculated based on the positive area per ovary. ELISA ELISA (Enzyme-Linked Immunosorbent Assay) was employed to assess the levels of anti-Müllerian hormone (AMH, Elabscience, China) in the ovarian culture medium. A 96-well plate coated with antibodies was incubated with ovarian culture medium for 2 h at room temperature. The reaction was recorded using a microplate reader to measure the absorbance, allowing for the determination of hormone concentrations in the culture medium. Iron assay A ferrous ion detection kit (Elabscience, China) was utilized to quantitatively measure the ferrous ion concentration in the ovaries following the manufacturer’s instructions. Ferrous ions in the samples bind to the probe, generating a substance with a strong absorption peak at 593 nm. The optical density values of this substance are linearly correlated with the ferrous ion concentration within a certain range. Detection was performed using a microplate reader. Total glutathione (T-GSH) and glutathione (GSH) levels The ovarian tissue’s intracellular GSH levels were assessed using the Total Glutathione (T-GSH) / Oxidized Glutathione (GSSG) assay kit (Elabscience, China). The assay employed the reduction of GSSG to GSH by glutathione reductase. GSH then reacted with the chromogenic substrate DTNB to produce GSSG and a yellow-colored TNB product. The quantity of total glutathione (GSSG + GSH) was determined by measuring the absorbance at 412 nm using a microplate reader. Additionally, to determine the GSSG content, the assay involved pretreating the samples to eliminate GSH, followed by the same reaction principle. The absorbance values obtained at 412 nm allowed for the calculation of both total glutathione and GSSG levels. RT-PCR According to the manufacturer’s protocol, TRIzol reagent was used to isolate and purify total RNA, followed by reverse transcription to generate cDNA. Quantitative real-time PCR using SYBR Green format on a 96-well optical plate was conducted for detection using a Roche quantitative PCR machine (Table [72]1). The mRNA expression levels of NANOS3, LHX8, NOBOX [[73]33], ACLS4, GPX4, TFRC, PTGS2, CHAC1 and NCOA4 were calculated using the formula 2^−ΔΔCt. The ratio of mRNA expression was calculated relative to the untreated control group after normalization to the housekeeping gene ACTIN. Table 1. List of qRT-PCR primers Gene Forward primer (5′-3′) Reverse primer (5′-3′) β-ACTIN GGAGATTACTGCCCTGGCTCCTA GACTCATCGTACTCCTGCTTGCTG LHX8 ACGGTAATGGGATTAGTGTGGAAGG GTTGTTGTCCTGAGCGAACTGTG NOBOX CTCTGGGTCCTGTTCAAACTCCTC GCCTTGTCCTTGTATATGCTGTCAC NANOS3 ACACTTCTGTCTACTGCTACACCAC CACCTGCTGCTGCTTCTCCTC GPX4 CTCCGAGTTCCTGGGCTTGTG CCGTCGATGTCCTTGGCTGAG ACSL4 ATTGGCTACTTACCTTTGGCTCATG GTACAATCACCCTTGCTTCCCTTC TFRC TGAAACTGGCTGAAACGGAGGAG AGGTCTGCCCAATATAAGCGAGATG PTGS2 TGGTGCCTGGTCTGATGATGTATG GTCTGCTGGTTTGGAATAGTTGCTC CHAC1 GTAAGAGCAGGGTAGCAGGGTTC CCGAGGCTTCTCTGGTCACAAC NCOA4 AGTTCCTTGTCAGAGTGGCTTATGG ACCCAGTCGGCAGTGTTAAAGG [74]Open in a new tab Western blotting Ovarian tissue was collected and processed for protein extraction using RIPA Lysis Buffer. The tissue samples were lysed using an ultrasonic disruptor, followed by centrifugation at 12,000 × g for 10 min to collect the supernatant. Subsequently, the extracted samples were diluted to a concentration of 1 µg/µl using 4× sample buffer and stored at -20 °C. The protein samples were boiled for 5 min and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skim milk at room temperature for 1 h, followed by overnight incubation with primary antibodies at 4 °C. After washing three times with TBST, the target protein membrane was incubated with secondary antibodies at room temperature for 1 h. Enhanced chemiluminescence (ECL, Biosharp, USA) were used for protein imaging and peroxidase activity detection. Quantitative analysis of grayscale values was performed using ImageJ software. RNA-sequencing (seq) analyses The RNA-Seq service was provided by BGI Genomics in Beijing. In summary, to ensure sample quality, BGI Genomics initially extracted RNA from ovarian tissue, enriched it using Oligo (dT) magnetic beads, followed by reverse transcription using N6 random primers, and finally sequenced it using the BGISEQ-500 platform. Subsequently, DESeq2 (version 1.4.5) was employed to conduct differential expression analysis, screening for genes with a Q value of ≤ 0.05. Following this, pathway enrichment analysis based on the KEGG database was performed for these identified genes. Statistics analysis All experiments were conducted with at least three replicates. Data is presented as mean ± standard deviation (SD) and analyzed using SPSS 26.0 software (IBM, Armonk, NY, USA). Intergroup comparisons were performed using analysis of variance (ANOVA) followed by LSD post hoc tests. A significance level of P < 0.05 was considered statistically significant. Statistical analyses were also conducted using GraphPad Prism 9.0 software. All statistical tests used were either ANOVA with Tukey’s post hoc test or the Student’s t-test. A significance level of P < 0.05 was considered statistically significant. Results Cisplatin induced excessive activation of follicles while upregulating the expression of ferroptosis-related genes in the in vitro POF model To investigate the effects of cisplatin on follicle in vitro POF model, the morphology of the follicle was observed using HE staining, and stages of follicle development were classified and quantified (Fig. [75]2A). Compared to the CTR group, the total number of follicles in the CIS group did not show significant changes. However, the CIS group showed a significant decrease in primordial follicles and an increase in secondary and antral follicles. (Fig. [76]2B). qRT-PCR analysis revealed significantly higher mRNA expression of Nanos3, Nobox, and Lhx8, follicular development-related genes, in the CIS group compared to the CTR groups (Fig. [77]2C). These findings suggested that cisplatin promoted the development of primordial follicles leading to excessive follicular activation in vitro POF model. Fig. 2. [78]Fig. 2 [79]Open in a new tab Cisplatin promoted excessive activation of follicles while inducing ferroptosis, leading to ovarian damage in vitro POF model. A Histological analysis of ovarian tissue after 4 days of in vitro culture stained with H&E. Scale bar = 100 μm (n = 10). B The number of follicles in two groups and the percentage of follicles at different stages in the two groups. C mRNA expression levels of Lhx8 (D), Nobox (E), and Nanos3 (F) were measured by qRT-PCR (n = 5). D Volcano plot analysis illustrates the quantity and upregulation/downregulation of expressed genes between the cisplatin and control groups. E Heat map analysis revealed the expression of ferroptosis-related genes after the addition of cisplatin. F mRNA expression levels of ACSL4, TFRC, NCOA4, CHAC1, PTSG2 detected using qRT-PCR (n = 5). *P < 0.05, **P < 0.01 and ***P < 0.001 To further explore the potential mechanisms underlying cisplatin-induced ovarian damage, we conducted RNA-seq analysis of mouse ovarian in vitro POF model. The volcano plot results revealed significant alterations in 1194 genes, among which 568 genes were upregulated, and 626 genes were downregulated (Fig. [80]2D). Heat map analysis revealed an upregulation in the expression of ferroptosis-related genes after the addition of cisplatin, suggesting that cisplatin-induced ovarian damage through the induction of ferroptosis (Fig. [81]2E). Subsequently, the mRNA expression of ACSL4, TFRC, NCOA4, CHAC1, and PTGS2, the ferroptosis-related genes, were validated, and these genes significantly increased in the CIS group compared to the CTR groups (Fig. [82]2F). These findings further underscored the strong link between cisplatin-induced ovarian damage and exacerbated ferroptosis. hUCMSCs alleviated the cisplatin-induced ovarian damage through inhibition of ferroptosis To confirm the therapeutic effect of hUCMSCs on cisplatin-induced ovarian injury, we used hUCMSCs co-cultured with ovaries in vitro POF model. Following treatment with erastin (ferroptosis inducer), the ovarian tissue displayed morphological shrinkage of follicles and an increase in arrested follicles, as observed through HE staining. After treatment with hUCMSCs, the ovarian tissue morphology was intact and the arrested follicles increased, as well as the healthy follicles significantly raised (Fig. [83]3A, B, C). Subsequently, Fe^2+ levels of ovarian tissue were assessed, and an elevated Fe^2+ was exhibited after exposure to both cisplatin and erastin, surpassing levels in the control group. Notably, hUCMSCs attenuated the increase in Fe^2+ induced by cisplatin (Fig. [84]3D). Quantitative analysis of total glutathione (T-GSH) and reduced glutathione (GSH) levels in ovarian tissue showed that the addition of cisplatin and erastin inhibited the expression of T-GSH and GSH. Significantly, HUCMSCs were effective in restoring this imbalance (Fig. [85]3E, F). The results of Prussian blue staining further confirmed the accumulation of iron in ovarian slices from the cisplatin and erastin groups, while this iron accumulation was mitigated after hUCMSCs treatment (Fig. [86]3G). These collective findings suggested that hUCMSCs alleviated cisplatin-induced damage to ovarian tissue by inhibiting ferroptosis. Fig. 3. [87]Fig. 3 [88]Open in a new tab Inhibition of cisplatin-Induced Ovarian Damage by hUCMSCs through Ferroptosis Suppression. A Histological analysis of ovarian tissue after 4 days of in vitro culture using H&E staining. Scale bar = 100 μm (n = 10). B Percentage of follicles at different developmental stages in four experimental groups. C Analysis of follicles in four experimental groups. D Quantitative assessment of intracellular ferrous ion levels in ovarian tissue (n = 6). E, F Total Glutathione (T-GSH) and reduced Glutathione (GSH) levels in ovarian tissue (n = 6). G Prussian blue staining of ovarian tissue in four experimental groups. Scale bar = 100 μm (n = 3) *P < 0.05, **P < 0.01 and ***P < 0.001 We examined the expression of special ferroptosis markers ACSL4 and GPX4 (Fig. [89]4A, Fig.[90]S1). The results indicated a significant increase in ACSL4 protein expression and a decrease in the expression of the ferroptosis-inhibiting GPX4 after the addition of cisplatin and ferroptosis inducer erastin (Fig. [91]4B, C). However, hUCMSCs decreased ACSL4 expression and increased GPX4 expression. Immunohistochemistry results also displayed increased expression of ACLS4 and reduced expression of GPX4 in ovarian tissues of the CIS and Erastin groups (Fig. [92]4D, E, F, G). This suggested that hUCMSCs repaired the cisplatin-induced POF through inhibition of ferroptosis. Fig. 4. [93]Fig. 4 [94]Open in a new tab hUCMSCs repaired ovarian damage by inhibiting ferroptosis. A Western blot of ACSL4 and GPX4 (Full-length blot are presented in Supplementary Figure). B, C Quantitative protein results analyzed (n = 4). D, E Immunohistochemistry of ACSL4 and quantitative analysis. Scale bar = 100 μm (n = 6). F, G Immunohistochemistry of GPX4 and quantitative analysis. Scale bar = 100 μm (n = 6). *P < 0.05, **P < 0.01 and ***P < 0.001 hUCMSCs rescued hormonal levels and reduced ovarian fibrosis by suppressing ferroptosis HE staining revealed that Fer-1 (ferroptosis inhibitor) exhibited effects similar to MSCs, showing improvement in ovarian tissue morphology and a reduction in atretic follicles (Fig. [95]5A). To assess the expression levels of AMH, we collected ovarian culture medium to examine the hormone secretion of follicles in the vitro system. AMH was significantly suppressed upon cisplatin administration. However, hUCMSCs and the Fer-1 could increase the expression levels of AMH (Fig. [96]5B). These results suggested that cisplatin leads to a decline in hormone expression in vitro tissue, whereas UCMSCs as well as Fer-1could rescue this process. Following this, we employed Masson’s trichrome staining to observe the effect of chemotherapy drugs on ovarian stromal fibrosis (Fig. [97]5C). Quantitative analysis of fibrosis revealed that there was notable disruption and increased fibrosis in the ovarian stroma after cisplatin administration (Fig. [98]5D). However, the ovarian stromal morphology exhibited signs of recovery treated with hUCMSCs and Fer-1, accompanied by a decrease in fibrosis levels. These findings indicated that hUCMSCs could ameliorate POF, recovery hormone levels, and reduce the degree of ovarian fibrosis. Fig. 5. [99]Fig. 5 [100]Open in a new tab Inhibiting ferroptosis reduced ovarian fibrosis and improved hormone levels. A Ovarian morphology under different conditions post in vitro culture using HE staining. Scale bar = 100 μm. B Assessment of hormone levels in ovarian culture medium (n = 6). C, D Evaluation of ovarian tissue fibrosis through MASSON staining (Blue represents collagen deposition. Scale bar = 100 μm (n = 10)). *P < 0.05, **P < 0.01 and ***P < 0.001 The expression of fibrosis-related factors COL-1 and α-SMA were assessed, and the result demonstrated that cisplatin significantly elevated the expression of ovarian tissue fibrosis factors, intensifying tissue fibrosis (Fig. [101]6A, Fig.[102]S2). However, hUCMSCs and Fer-1 reduced the expression of COL-1 and α-SMA (Fig. [103]6B, C). Immunohistochemistry (IHC) revealed similar findings, with increased expression of COL-1 and α-SMA in the CIS group, whereas hUCMSCs or Fer-1 inhibited the expression of the cisplatin-induced related factors (Fig. [104]6D, E, F, G and [105]7). These results indicated that hUCMSCs could alleviate the degree of ovarian tissue fibrosis by inhibiting ferroptosis in vitro POF model. Fig. 6. [106]Fig. 6 [107]Open in a new tab hUCMSCs reduced ovarian fibrosis by inhibiting ferroptosis. A Western blot of COL-1 and α-SMA (Full-length blot are presented in Supplementary Figure). B, C Quantitative protein results analyzed (n = 3). D, E Immunohistochemistry of COL-1 and quantitative analysis. Scale bar = 100 μm (n = 6). F, G Immunohistochemistry of α-SMA and quantitative analysis. Scale bar = 100 μm (n = 6). *P < 0.05, **P < 0.01 and ***P < 0.001 Fig. 7. [108]Fig. 7 [109]Open in a new tab Cisplatin suppresses GPX4 activity, leading to increased oxidative stress and ROS production, which in turn promotes lipid peroxidation. This lipid peroxidation triggers fibrosis through the upregulation of α-SMA and COL-I and induces ferroptosis through the activation of ACSL4 and TFRC. hUC-MSCs mitigate these effects by reducing oxidative stress, thereby reducing lipid peroxidation and preventing the subsequent fibrosis and ferroptosis induced by cisplatin([110]www.figdraw.com) Discussion Chemotherapeutic agents are predominantly utilized to disrupt the cancer cells during cancer treatment. Unfortunately, they also induce damage to normal tissues, with ovarian impairment being a common side effect in females undergoing chemotherapy [[111]34]. Long-term chemotherapy-induced ovarian damage often results in reduced ovarian reserve, infertility, and ovarian atrophy [[112]35]. This study employed a co-culture model in vitro to investigate the effects of cisplatin on ovarian function and validate the reparative role of UCMSCs in the in vitro ovarian culture system, shedding light on their specific mechanism and therapeutic potential for POF. Ferroptosis, a non-apoptotic form of cell death, is characterized by an accumulation of intracellular reactive oxygen species (ROS) and is implicated in various pathological processes such as cancer, neurodegeneration, and tissue injuries [[113]36]. Chemotherapy can induce depletion of glutathione (GSH) and inactivation of glutathione peroxidase 4 (GPX4), leading to excessive accumulation of reactive oxygen species (ROS) and subsequent ovarian damage through ferroptosis [[114]37]. GSH, as one of the most abundant non-protein thiol groups in cells, mainly binds to cisplatin in the cytoplasm to form Pt-GSH complexes. The depletion of GSH, combined with GPX4 inactivation, represents a potential mechanism underlying cisplatin-induced ferroptosis [[115]38, [116]39]. Our research findings also demonstrate that both cisplatin and erastin could decrease the expression of GSH in vitro ovarian culture system, leading to a reduction in GPX4 expression, further causing P through ferroptosis. Fibrosis is a consequence of tissue repair responses, becoming difficult to restore after tissue injury, chemotherapy also has a certain impact on tissue fibrosis [[117]40–[118]42]. After treatment with cisplatin in the in vitro system, there was a significant reduction in the number of primary follicles in the ovaries, and the morphology of mature follicles became abnormal. Subsequent examinations further revealed that chemotherapy markedly exacerbated ovarian fibrosis, leading to ovarian damage. Overall, chemotherapy drugs promoted ovarian cell apoptosis, reduced ovarian receptivity, induced ovarian fibrosis, and led to ovarian damage (morphological abnormalities and functional impairment) [[119]43, [120]44]. The intricate relationship between ferroptosis and fibrosis is not yet fully understood, but existing perspectives suggest that ferroptosis exacerbates tissue fibrosis [[121]22, [122]45]. Targeting ferroptosis with ferroptosis inhibitors, which reduce the expression of GPX4, can alleviate ROS accumulation and mitochondrial damage, further reducing fibrosis [[123]46, [124]47]. Our experiments confirm that cisplatin, when applied in an in vitro ovarian culture system, reduces the expression of GPX4 by activating the ferroptosis pathway and upregulates ACSL4 expression, thereby increasing the expression of α-SMA and COL-1, exacerbating the degree of ovarian fibrosis. This result clarifies the effects and mechanisms of chemotherapy drugs on ovarian tissue and provides new insights into the safety of drug usage for cancer patients. hUCMSCs, a widely accessible and highly promising type of stem cell, have been demonstrated to be effective in alleviating tissue injuries in numerous studies [[125]48, [126]49]. Particularly noteworthy is MSCs’ ability to mitigate cellular senescence and apoptosis, making them extensively used in the treatment of ovarian damage [[127]50, [128]51]. Prior research indicates that inhibiting MSCs’ ferroptosis contributes to enhancing their reparative effects, MSCs can also improve tissue damage by inhibiting ferroptosis. MSC therapy significantly reduces abnormalities in iron metabolism, GSH inactivation, decreased levels of GPX4, and increased lipid peroxidation [[129]52–[130]54]. In our study, UCMSCs exhibited similar effects to Fer-1, reducing the elevation of ferrous ions induced by cisplatin, increasing the GSH content, thereby enhancing GPX4 expression, and decreasing ACSL4 expression. This suggests that hUCMSCs alleviate cisplatin-induced ovarian damage by inhibiting ferroptosis. Subsequently, we found that hUCMSCs alleviated the degree of ovarian fibrosis by inhibiting ferroptosis, leading to increased expression of ovarian AMH, and reduced expression levels of COL-1 and α-SMA. Overall, UCMSCs mitigate cisplatin-induced ovarian damage by inhibiting the ferroptosis pathway, decreasing the extent of ovarian fibrosis, and improving ovarian hormone levels. Currently, techniques for in vitro culture of ovarian tissue or isolated follicles or oocytes have gradually matured. However, culturing isolated follicles, oocytes, or ovarian tissue blocks only partially simulates the different interactions between follicles and other cell types within the complete structure. This study employed whole ovarian in vitro culture, which better mimics the physiological structure in vivo compared to other culture methods. This approach maintains a more intact ovarian structure, enabling better observation of morphological and functional changes in the ovaries [[131]10, [132]30, [133]55, [134]56]. We have treated ovarian tissue with cisplatin in vitro, which eliminates interference from other factors in vivo and allows for faster, more controllable, and adjustable effects. Under the influence of cisplatin, ovarian follicles are excessively activated, leading to depletion of primordial follicles. Moreover, there is an increase in mRNA expression of follicular development-related genes such as LHX8, NOBOX, and NANOS3, similar to the results observed in vivo [[135]57, [136]58]. Interestingly, we observed a significant improvement in cisplatin-induced ovarian damage after the application of hUCMSCs for repair. After repair by hUCMSCs, the number of blocked ovarian follicles decreased and the morphology of the follicles became more intact. In addition, the excessive depletion of primordial follicles was alleviated. Notably, hUCMSC intervention also reduced the degree of ovarian fibrosis, restored hormone levels, and improved tissue structure and function of damaged ovaries. These findings highlight the crucial role of MSCs in repairing ovarian damage, which is essential for preserving fertility in POF patients following chemotherapy. To further validate the role of MSCs in clinical treatment, it is essential to comprehensively understand their mechanisms of action [[137]59]. MSCs might exert a positive impact on tissue injury through their immunomodulatory effects, regulating immune cell activity, and influencing cytokine release, thereby suppressing inflammatory reactions, and reducing tissue inflammatory damage [[138]60]. The antioxidant properties of MSCs might play a crucial role in tissue restoration by reducing oxidative damage [[139]61, [140]62]. Additionally, MSCs may indirectly participate in the repair process of damaged tissues by promoting cell proliferation and differentiation, releasing growth factors, and regulating cellular interactions through paracrine mechanisms, thereby facilitating ovarian tissue follicle activation [[141]63, [142]64]. The specific mechanism by which MSCs repair ovarian damage is likely through exosomes, and the specific molecular regulatory mechanisms within exosomes are still worth exploring. This will be the focus of our future research [[143]65, [144]66]. These multiple mechanisms collectively contribute to the favorable performance of MSCs in repairing tissue injury. Nevertheless, further in-depth preclinical research and clinical trials are needed to verify the safety and efficacy of MSCs as a therapeutic approach. A detailed understanding of their performance in different types and degrees of ovarian injury, as well as their combined effects with other therapeutic modalities, is needed. In conclusion, the broad mechanisms of action of MSCs provide a solid basis for their application in repairing ovarian injury. Further extensive research is warranted to fully explore their therapeutic potential and provide robust support for clinical applications. Conclusion In conclusion, we have confirmed that cisplatin induces ovarian damage in vitro POF model via ferroptosis. hUCMSCs alleviate ovarian injury by suppressing ferroptosis, promoting follicular development, and reducing ovarian fibrosis levels. Overall, our research explores that hUCMSCs could be a potential therapeutic strategy for treating chemotherapy-induced POF by inhibiting the ferroptosis pathway and suppressing fibrosis, thus offering a promising approach for restoring POF and fertility in cancer patients. Electronic supplementary material Below is the link to the electronic supplementary material. [145]Supplementary Material 1^ (970.3KB, docx) Acknowledgements