3D hUC-MSC spheroids exhibit superior resistance to autophagy and apoptosis of granulosa cells in POF rat model

In brief This study reveals that orthotopic transplantation of 3D hUC-MSC spheroids is more effective than monolayer-cultured hUC-MSCs in improving POF and distinctly reducing oxidative stress through the paracrine effect, thereby preventing apoptosis and autophagy of GCs. Abstract Premature ovarian failure (POF) is a common reproductive disease in women younger than 40 years old, and studies have demonstrated that the application of human umbilical cord mesenchymal stem cells (hUC-MSCs) is a promising therapy strategy for POF. Given the previously established therapeutic advantages of 3D MSC spheroids, and to evaluate their effectiveness, both 3D hUC-MSC spheroids and monolayer-cultured hUC-MSCs were employed to treat a cyclophosphamide-induced POF rat model through orthotopic transplantation. The effects of these two forms on POF were subsequently assessed by examining apoptosis, autophagy, and oxidative damage in ovarian granulosa cells (GCs). The results indicated that hUC-MSC spheroids exhibited superior treatment effects on resisting autophagy, apoptosis, and oxidative damage in GCs compared to monolayer-cultured hUC-MSCs. To further elucidate the impact of hUC-MSC spheroids in vitro, a H2O2-induced KGN cells model was established and co-cultured with both forms of hUC-MSCs. As expected, the hUC-MSC spheroids also exhibited superior effects in resisting apoptosis and autophagy caused by oxidative damage. Therefore, this study demonstrates that 3D hUC-MSC spheroids have potential advantages in POF therapy; however, the detailed mechanisms need to be further investigated. Furthermore, this study will provide a reference for the clinical treatment strategy of POF.


Introduction
POF is a disease of the female reproductive system that affects approximately 2% of women under 40, resulting in amenorrhea, low estrogen levels, and follicle failure (Chon et al. 2021).Infertility is the most direct consequence of POF, while other complications such as cardiovascular disease, osteoporosis, and sexual dysfunction are also associated with POF (Liu et al. 2021, Wang et al. 2022).Therefore, POF has a significant effect on the reproductive health and quality of life of women.Current research suggests that the primary cause of POF is low ovarian reserve function due to atresia of the dominant follicles and failure of the primordial follicles in the ovary due to oxidative stress (Sun et al. 2018, Asadi et al. 2022, Jiang et al. 2019).GCs play a crucial role in follicular development and maturation by producing estradiol and other maturation-promoting factors (Zhu et al. 2022).Studies have revealed that apoptosis and excessive autophagy of GCs caused by oxidative stress are the primary causes of follicular dysfunction and POF occurrence (Shen et al. 2018, Xu et al. 2022).
Autophagy is a physiological process that helps in maintaining cellular homeostasis; however, persistent oxidative stress can lead to excessive autophagy, resulting in cell death (Zhou et al. 2019).Normal autophagy is essential for the selection of dominant follicles and follicle development in GCs, whereas excessive autophagy can cause follicle atresia (Liu et al. 2023, Yin et al. 2020).To maintain the homeostasis of GCs, the antioxidant system composed of Glutathione (GSH), Super Oxide Dismutase (SOD), and Catalase (CAT) plays a vital role in resisting oxidative stress.Therefore, to improve a variety of cytokines, such as VEGF, EGF, HGF, IGF, TNF-α, NGF, and TrkA, which regulate the immune system and repair damaged tissues (Li et al. 2021).Multiple lines of clinical transplantation trials have confirmed the effectiveness of hUC-MSCs in the treatment of a variety of diseases, demonstrating their promising clinical applications (Hernandez et al. 2021, Litvinova et al. 2022).Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) are superior in the treatment of several diseases including POF (Umer et al. 2023).Additionally, hUC-MSCs have a high proliferation ability, powerful secretion ability, low immunogenicity, and few ethical concerns due to discarded perinatal tissue (Huang et al. 2016, Umer et al. 2023, Hai et al. 2022).
Therefore, the application of hUC-MSCs is considered one of the optimal strategies for POF treatment.However, studies have shown that MSCs transplanted intravenously have a very low success rate for ovarian tissue (Kim et al. 2020, Belmadi et al. 2015).
Orthotopic transplantation can increase the number of hUC-MSCs in ovarian tissue treatment, thereby improving the therapeutic effect (Huang et al. 2022).Numerous studies have demonstrated that 3D MSCs spheroids can maintain their survival rate and paracrine capacity in injury tissue while also simulating the real situation in tissue more accurately (Xu et al. 2016, Jiang et al. 2019, Yuan et al. 2022).Therefore, we hypothesized that orthotopic transplantation of 3D hUC-MSCs spheroids could increase the therapeutic effect on POF.
In this study, the therapeutic effects of monolayer cultured hUC-MSCs and 3D hUC-MSCs spheroids on the CTX-induced POF rat model were compared by evaluating the apoptosis and autophagy of GCs.Studies including ours have shown that CTX-induced POF is due to oxidative damage of GCs (Chen et al. 2022, Dai et al. 2023).
Subsequently, the levels of autophagy and apoptosis and the antioxidant effects of two forms of hUC-MSCs on H 2 O 2 -induced KGN cells were compared in vitro to clarify the therapeutic effects of hUC-MSCs on POF (Shen et al. 2018, Shen et al. 2017).This study will provide a novel strategy and a reference for the clinical application of hUC-MSCs in the treatment of POF.

Isolation and identification of hUC-MSCs
The umbilical cords of full-term neonates were collected after receiving ethical approval from the Ethical Review Committee of Ningxia Medical University and after the cord donors and their families signed the informed consent forms.The cord tissue was sterilized with 75% alcohol for 30 sec, placed in a PBS solution, and transferred within 1 h to a clean bench.The redundant fatty and blood vessels were removed from the tissue and dissected into 0.5 cm×0.5 cm pieces before being cultured in a sterile 10 cm plastic petri dish with 10 mL of low glucose Dulbecco's modified Eagle's medium 7 Kit (BD Biosciences, San Jose, CA) on a C6 flow cytometer (BD Biosciences, San Jose, CA) at the third passage.Briefly, 2×10 6 hUC-MSCs were collected and resuspended with 500 μL of PBS (containing 3% FBS, PBSF).Then, 5 µL (10 µg/µL) of human monoclonal antibodies against positive markers (CD44, CD73, CD90, and CD105) and hematopoietic markers (including CD34, CD45, CD14, CD19, and HLA-DR) were incubated for 30 min at room temperature, according to the manufacturer's instructions.
Unbound antibodies were washed with PBS, and the cells were resuspended in 500 µL of PBSF.Then, the labeled MSCs were assayed by FACS Aria II (BD Biosciences), and 10 4 events were recorded, the data were analyzed by the built-in software of the instrument.The details have been described in our previous study (Fu et al. 2020).

Animal experiments
A total of 20 inbred female specific pathogen-free (SPF) Sprague-Dawley (SD) rats five weeks (mean body weight 180 ± 20 g) were obtained from the Experimental Animal Center of Ningxia Medical University.All handling and animal care procedures were performed by the guidelines of the National Institutes of Health of China and were approved by the Medical Ethics Committee of Ningxia Medical University .
All animals were given ad libitum access to food and sterile water under a controlled temperature of 22 ± 2 °C and a 12 h light/dark cycle.The rats were randomly divided into the Control (Control, n = 5) and POF (POF, n = 15) groups.The estrus cycle was detected by vaginal exfoliated cell smear, and the estrus cycle of 96-120 h was selected for the subsequent establishment of animal models.The rats of the POF group were intraperitoneally injected with CTX (50 mg/kg/day for the first day, followed by 8 mg/kg/day for 14 days), while the rats of Control were injected with an equal amount of saline (Dai et al., 2023).After successfully establishing the POF group, rats were randomly divided into three groups (n = 5 in each group): the POF+Saline group (for control of treatment groups), the POF+MSCs (M) group (for transplanting monolayer cultured hUC-MSCs suspension), and the POF+MSCs (S) group (for transplanting 3Dcultured hUC-MSCs spheroids), while the normal control group continued to be fed.
After the 5 th passage, hUC-MSCs were collected and washed three times with saline.
At 48 days after the orthotopic transplantation of MSCs, rats were euthanized, and ovaries were collected for ovarian parameters and function evaluations.Based on 9 consecutive days of estrus cycle measurement, blood was collected from the tail vein during anoestrum for hormone measurements.

Preparation of 3D hUC-MSCs spheroids and orthotopic transplantation
The obtained third-generation hUC-MSCs were resuspended in fresh medium and the concentration was adjusted to 1×10 6 cells/mL.25 μL of the cell suspension was evenly dripped onto the lid of a 10 cm sterile Petri dish, gently turned over, and then the dish was inverted onto a Petri dish with PBS and gently placed into the incubator for 48 h.
The prepared 3D hUC-MSCs spheroids were collected for subsequent co-culture experiments (Fig. S1A).After routine disinfection, the skin and muscle were cut from the dorsal costovertebral angle, and the ovaries were exposed in a sterile environment.
The ovaries and fallopian tubes were removed, and the monolayer cultured hUC-MSCs suspension or 3D-cultured hUC-MSCs spheroids were injected steadily into the ovary.
The ovary was sutured with an ophthalmic needle (1/2 arc, 8×20, Shanghai Medical Suture Needle Co. LTD.), and the ovary and fallopian tube were returned slowly to the abdominal cavity, followed by the suturing of the muscles and skin.The rats in the POF+MSCs (M) group were injected with monolayer cultured hUC-MSCs suspension (1×10 6 cells/20 μL) by orthotopic transplantation into the ovaries, the rats in the POF+MSCs (S) group were injected with 3D-cultured hUC-MSCs spheroids (equal with 10 6 cells), whereas the Control and POF+Saline groups were injected with 20 μL of saline.Subsequent assays were detected after 48 days of hUC-MSCs transplantation (Fig. S1B).

Histology evaluation and follicle counting
The ovaries of rats in each group were firstly removed and fixed with 4% paraformaldehyde in a refrigerator at 4 °C for 24 h, followed by gradient alcohol dehydration and embedding with paraffin.Secondly, the ovaries were serially sectioned at a thickness of 5 μm, and multiple sections were selected at five intervals for hematoxylin and eosin (H&E) staining.Finally, ovarian morphology was observed and photographed using microphotographic equipment (Tissue Nostic, Austria).The total number of primordial follicles, primary follicles, secondary follicles, mature follicles, and atretic follicles were counted, avoiding double counting by different markers.The classification of follicular stages was described in our previous study (Fu et al. 2020).
Primordial follicles (PrF) were defined as an oocyte surrounded by a layer of squamous instructions.The sections were incubated with the primary antibody overnight at 4 °C, washed with PBS, incubated with horseradish peroxidase-labeled secondary antibody for 2 h, and then washed with PBS.After staining with diaminobenzidine (DAB), the color development reaction was terminated by adding water, the nuclei were stained with hematoxylin for 2 min, and the section was sealed with neutral resin.After drying, the positive cells were photographed with photomicrograph equipment (Tissue Gnostics, Austria).CYP19A1 (A12684) was purchased from ABclonal Technology Co., Ltd., China; 4-HNE (4-Hydroxynonenal) was purchased from Bioss Biotech Co. Ltd., China.All primary antibodies were diluted at 1:200.

Determination of serum hormones
Follicle-stimulating hormone (FSH), estradiol (E2), and luteinizing hormone (LH) levels were determined according to the Elabscience ELISA kit instructions.The serum in each group of rats was diluted 1:10, 96-well plates containing antibodies from the kit were removed, and the diluted serum was added gently and accurately and co-incubated in a thermostat at 37 °C for 2 h.After completion of the reaction, a termination solution was added, and serum hormone levels were determined by using an enzyme labeling instrument (Thermo Fisher Scientific, USA).Three serum samples in each group were tested.

Cell culture and treatment
The human ovarian granulosa cell line KGN was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.KGN was cultured in DMEM/F12 medium supplemented with 10% FBS and 1% penicillin/streptomycin incubated at 37 °C and 5% CO 2 .The KGN cells were planted in a six-well plate at a density of 2.5×10 5 cells/well and cultured for 24 h, and 3 or 6 hUC-MSCs spheroids or an equal number of monolayer cultured hUC-MSCs were co-cultured with KGN cells for 48 h in a transwell system.
Four hours before KGN cells collection, H 2 O 2 was supplemented at the working concentration of 100 μM.Finally, the KGN cells were used for subsequent determination (Shen et al. 2018, Shen et al. 2017).

Isolation, culture, and characterization of GCs
Following treatment with hUC-MSCs, female 3-week SD rats were anesthetized and euthanized to harvest their ovaries.Preovulatory follicles within the ovaries were subsequently punctured under a stereomicroscope to isolate GCs.The collected cells were then washed and cultured in a complete DMEM/F12 medium (VivaCell, Shanghai) supplemented with 10% fetal bovine serum (Gibco, USA) at 37 °C and 5% CO 2 .Upon subculture, purification, and characterization, the primary granulosa cells (GCs) were utilized for subsequent experimentation (Fig. S2A and B).
Briefly, tissue and cell samples were homogenated with an ultrasonic crusher and then centrifuged at 13,000 rpm/min for 15 min at 4 °C to obtain the supernatant.
Subsequently, the corresponding reagents were added and mixed with the supernatant according to the manufacturer's instructions.In terms of the detection principle, the reaction of MDA with thiobarbituric acid showed a red color and the absorbance at 532 nm was detected; the reaction of GSH with dithio-dinitrobenzoic acid (DTNB) was yellow and the absorbance at 405 nm was detected; The protein concentration was detected by the BCA protein assay kit.Our previous study has described the detailed procedures (Xu et al. 2023).The cell viability assay kit (CCK-8) (Beyond Biotech, Shanghai, China) was used according to the manufacturer's instructions.Briefly, KGN cells were seeded at a density of 8000 cells/well in a 96-well plate for 24 h, serial concentrations of H 2 O 2 were added, and the cells were cultured at 37 °C, 5% CO 2 for 4 h before being incubated with 10% (v/v) kit reagent for 2 h.The absorbance of each well was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, USA).The mean of three independent tests was calculated to determine the effect of H 2 O 2 on viability.

Western blotting
Ovary tissues or KGN cells were treated on ice for 30 min with radioimmunoprecipitation assay (RIPA) lysis buffer containing the protease inhibitor.
After homogenization, the protein extraction solution was harvested by centrifuging at 12,000 rpm/min for 15 min at 4 °C.The total protein concentration of each sample was determined using the BCA protein assay kit (KeyGEN Biotech Co., LTD, Jiangsu).
After denaturation at 100 °C for 10 min, 40 μg of each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 100 V.The separated proteins were then transferred to PVDF membranes by immunoblotting.The PVDF membrane was blocked for 30 min with 0.05 g/mL defatted milk in PBS containing 1% (v/v) Tween 20 (PBST).After washing with PBST, the primary antibody was incubated overnight at 4 °C, followed by a 2 h incubation with the secondary antibody.Finally, the blotting was detected using an enhanced chemiluminescence (ECL) kit (KeyGEN Biotech Co., LTD, Jiangsu) on a fluorescence detection instrument (Bio-Rad, USA).All primary antibodies were diluted at 1:1000, whose sources were provided in the supplementary material.Representative blotting images were selected from at least three independent replicate experiments.

KI67 immunofluorescence
The KGN cells (1.5×10 4 ) were seeded into a well of the 24-well plate which had been pre-positioned with cell crawlers (round coverslip), and then co-culture with hUC-MSCs spheroids and treated with H 2 O 2 .After co-culturing, the medium was removed and 4% paraformaldehyde was treated for 10 min.Then, the cells were washed three times with PBS containing 0.5% Triton and blocked with 10% goat serum for 30 min, and KI67 primary antibody diluted by 1:200 was added and incubated at 37 °C for 2h, then washed with PBS and replaced with 1% BSA diluted fluorescent secondary antibody, incubated at 37 °C for 1 h and stained with DAPI for 10 min.After rinsing with PBS and sealing with an anti-fluorescence quenching reagent, the images were captured by using a confocal microscope (Nikon, Japan).

Statistical Analysis
ImageJ software (National Institutes of Health) was used to quantify the target protein and the internal reference protein to quantify the protein expression.All data were represented as the mean ± standard deviation (SD) using GraphPad Prism 8.0 software.Data was tested for normality before analysis of variance (ANOVA) or t-tests using GraphPad Prism 8.0 software.Various factors were compared using two-way ANOVA, and multiple samples were compared using one-way ANOVA with the minimum significant difference (LSD) method.P value less than 0.05 was considered statistically significant.

Isolation, culture, and identification of hUC-MSCs
The umbilical cord tissue was sectioned and cultured in an incubator (Fig. 1A).After one week, spindle-shaped, polygonal, or stellate cells appeared at the periphery of tissue pieces.The cells were passaged to the third generation, and their morphology was identified; the results showed long spindle-shaped and monolayer vortex-shaped cells (Fig. 1B, a).After adipogenic, osteogenic, and chondrogenic inducing differentiation for 21 days, hUC-MSCs have the potential for osteogenesis, chondrogenesis, and adipogenesis differentiation, which were identified by specific dyes.(Fig. 1B, b-d), respectively.The surface marker expression of hUC-MSCs at passage three was identified by flow cytometry.The results showed that more than 98% of hUC-MSCs expressed CD 44, CD 90, CD 73, and CD 105, but none expressed the hematopoietic markers of the kit (Fig. 1C).These results suggested that the isolated cells conformed to the standard of the International Society for Cell Therapy (ISCT), and could be used for subsequent studies.The weight gain of rats in the POF group was significantly slower than that of the Control group during the modeling period, whereas the weight gain of the hUC-MSCs orthotopic transplantation groups was significantly greater than that of the POF+Saline group (Fig. 2A and B).Subsequently, the ovarian organ coefficient results showed that the POF+Saline group was significantly lower than the Control group, whereas the POF+MSCs (M) and POF+MSCs (S) groups were restored considerably in comparison to the POF+Saline group (Fig. 2C).Compared to the Control group, ovarian histology revealed that the number of primordial follicles, primary follicles, and secondary follicles were substantially reduced in the POF+Saline group, while the number of atretic follicles was increased.After hUC-MSCs transplantation, the number of follicles at different stages was restored in the POF+MSCs (M) and POF+MSCs (S) groups compared to the POF+Saline and Control groups (Fig. 2D and E).These results indicated that two forms of hUC-MSCs transplantation could significantly improve the POF rat model.

hUC-MSCs improved ovarian function in the POF rat model
The estrus cycle was evaluated by staining the vaginal smears in rats, and the results showed the rats in the POF+Saline group had a longer diestrus period and a shorter estrus period than rats in the Control group.After hUC-MSCs transplantation, a significant improvement was observed in the POF+MSCs (S) group, and the administration of hUC-MSCs (S) effectively ameliorated the disruption of the estrous cycle induced by CTX, whereas no significant improvement in the POF+MSCs (M) group compared to the POF+Saline group (Fig. 3A).Additionally, the levels of FSH (Fig. 3B), E2 (Fig. 3C), and LH (Fig. 3D) in the POF+MSCs (M) and POF+MSCs (S) groups were significantly improved when compared to the POF+Saline group.
Therefore, these results suggested that the efficacy of hUC-MSCs spheroids orthotopic transplantation was superior to that of monolayer cultured hUC-MSCs suspension.

hUC-MSCs ameliorated apoptosis, autophagy, and oxidative stress in ovarian tissues of the POF rat model
Numerous studies showed that MSCs improved POF models by protecting the ovary from oxidative stress, apoptosis, and autophagy (Lu et al. 2019, Yin et al. 2018, Lu et al. 2020).In this study, we also compared the effects of two forms of hUC-MSCs on apoptosis, autophagy, and oxidative stress in ovaries after orthotopic transplantation.
Then, the expression of autophagy key proteins (P62, ATG5, and LC3A/B), apoptosis key proteins (Bcl-2 and Bax), and proliferation-associated protein (PCNA) were detected.The results showed that P62, Bcl-2, and PCNA were increased; ATG5 and LC3A/B were decreased in POF+MSCs (M) and POF+MSCs (S) groups, which compared to the POF+Saline group, however, the expression of Bax was only decreased in POF+MSCs (S) group (Fig. 3E and F).Additionally, the immunohistochemistry results showed that CYP19A1 (functional marker of GCs), was significantly increased and 4-HNE (lipid peroxidation marker) was decreased in the POF+MSCs (M) and POF+MSCs (S) groups, which compared to the POF+Saline group (Fig. 3G and Fig. S2D).The levels of oxidative stress indicators MDA (Fig. 3H) and GSH (Fig. 3I) in ovarian tissues of each group were compared, and the results showed that they were significantly improved after hUC-MSCs transplantation in the POF+MSCs (M) and POF+MSCs (S) groups, compared to the POF+Saline group.These findings indicated that hUC-MSCs orthotopic transplantation could ameliorate oxidative stress levels and inhibit autophagy and apoptosis in the ovary.Additionally, the expression of autophagy and apoptosis-related proteins in ovaries was examined by immunohistochemistry.The results showed that P62 and Bcl-2 were increased; LC3B and Bax were decreased in POF+MSCs (M) and the POF+MSCs (S) groups, which compared to the POF+Saline group (Fig. 4A, B and Fig. S2E).Interestingly, these results revealed that the improvements were mainly focused on the ovary's granulosa cell layer.Therefore, we hypothesized that hUC-MSCs could reduce the oxidative stress level of the ovary and inhibit autophagy and apoptosis of GCs, thereby restoring ovarian function and improving POF rats.

hUC-MSCs spheroids were superior to hUC-MSCs suspension in inhibiting oxidative damage in KGN cells
Studies have demonstrated that 3D MSCs spheroids have the superior paracrine ability and can better mimic the state of MSCs in the tissue after orthotopic transplantation (Xu et al. 2016, Remuzzi et al. 2020, Yue et al. 2016).In the present study, the coculture system of hUC-MSCs spheroids and H 2 O 2 -induced KGN cells was used to investigate the underlying mechanism.Firstly, KGN cells were treated with H 2 O 2 (50, 100, and 200 µM) for 4 h for optimal exposure conditions.The results showed that the  5A), and the Western blotting result showed that the expression of autophagy protein LC3A/B was significantly increased by 100 μM of H 2 O 2 (Fig. 5B).Therefore, the optimized condition of 100 μM H 2 O 2 for 4 h was used for subsequent experiments.To evaluate the recovery of H 2 O 2 -induced KGN cells by hUC-MSCs (M) and hUC-MSCs (S), a transwell co-culture system was established (Fig. 5C).The improvement was evaluated using the protein expression of LC3A/B, and the results showed that hUC-MSCs (S) significantly reduced the expression of LC3A/B, however, the equivalent amount of hUC-MSCs (M) did not significantly reduced it (Fig. 5D-F).This result suggested that the effect of hUC-MSCs spheroids was superior to the equal number of monolayer cultured hUC-MSCs in inhibiting H 2 O 2 -induced autophagy in KGN cells.

hUC-MSCs spheroids reduced H 2 O 2 -induced autophagy and apoptosis and improved the function and proliferation of KGN cells.
To further confirm the effect of hUC-MSCs spheroids on H 2 O 2 -induced GCs or KGN cells, they were co-cultured with hUC-MSCs spheroids and were collected for analysis.
In the H 2 O 2 +MSCs (S) group, the expression of autophagy-related proteins (P62, ATG5, and LC3A/B), apoptosis-related proteins (Bax and Bcl-2), and granulosa cell functionrelated protein (FSHR) was significantly improved compared to the H 2 O 2 -induced group (Fig. 5G-H).In addition, to further verify this result, primary granulosa cells of rats were isolated and co-cultured with hUC-MSCs spheroids, and then the expression of autophagy-related proteins (ATG5 and LC3A/B) and apoptosis-related proteins (Bax and Bcl-2) was detected, the results were consistent with those on KGN cells (Fig. S2C).
Subsequently, the result of immunofluorescence showed that the expression of the proliferation-related protein (KI67) was also significantly restored in the H 2 O 2 +MSCs (S) group, compared to the H 2 O 2 -exposed group (Fig. 5I and Fig. S2F).Additionally, the result of oxidative stress levels showed that the MDA level in the H 2 O 2 +MSCs (S) group was lower than that in the H 2 O 2 group (Fig. 5J), and the GSH level was higher than that in the H 2 O 2 group (Fig. 5K).These results suggested that hUC-MSCs spheroids could ameliorate the autophagy and apoptosis of KGN cells caused by oxidative damage.

Discussion
Numerous studies have demonstrated that hUC-MSCs can significantly improve ovarian function in various POF animal models due to their low immunogenicity (Zhao et al. 2020, Yang et al. 2019, Deng et al. 2021).The main disadvantage of transvenous transplantation is that the pulmonary vascular system will clear most of the MSCs, resulting in a decrease in the number of MSCs reaching the injured site, making it difficult to achieve a better therapeutic effect.Thus, transvenous transplantation may reduce the planting efficiency of MSCs in the injured ovary.In addition, the studies have demonstrated that orthotopic transplantation of hUC-MSCs could effectively improve ovarian function (Yan et al. 2020, Ding et al. 2018, Igboeli et al. 2020).These studies suggested that the feasibility of hUC-MSCs improves POF through orthotopic transplantation in clinical practice.
In the present study, we compared the therapeutic effects of monolayer cultured hUC-MSCs suspension and 3D hUC-MSCs spheroids by orthotopic transplantation in a POF rat model, the result showed the bodyweight gain was differential in four groups (Control, POF+Saline, POF+MSCs (M) and POF+MSCs (S)), this may be due to the toxicity of CTX in rats and MSCs can restore the toxic effect (Mo et al. 2024).In addition, the results indicated that both cell culture methods had significant effects in restoring ovarian function, but hUC-MSCs spheroids exhibited a superior therapeutic effect than monolayer hUC-MSCs suspension.Additionally, it has been shown that 3D MSCs spheroids are more effective in treating renal ischemia-reperfusion injury (Zhao et al. 2016) and ischemic stroke disease models (Li et al. 2021).An increasing number of studies have demonstrated that compared to the monolayer cultured MSCs suspension, the 3D MSCs spheroids have a higher survival rate and greater paracrine capacity, as well as superior stemness maintenance, anti-aging, and anti-inflammation abilities, and promote angiogenesis and tissue repair (Bhang et al. 2012, Guo et al. 2014, Bartosh et al. 2013, Domnina et al. 2018).Similar to our findings, Deng et al. demonstrated that MSCs spheroids had a better therapeutic effect on spinal cord injury in mice than monolayer-cultured MSCs (Deng et al. 2021).Cao et al. confirmed that 3D-cultured MSCs generate more exosomes than monolayer-cultured MSCs (Cao et al. 2020).Similar to ours, these results also confirmed that MSCs spheroids might have more beneficial effects in treating diseases.Additionally, orthotopic transplantation of MSCs combined with collagen scaffolds restored the ovarian function of mice in GCs, primary follicles, secondary follicles, and antral follicle atresia (Su et al. 2016), which is comparable to our results.Notably, orthotopic transplantation of MSCs significantly reduced the autophagy and apoptosis in POF ovarian tissue and alleviated oxidative stress, with GCs reflecting these changes the most.Subsequently, the co-culture system of hUC-MSCs spheroids and H 2 O 2 -induced KGN cells was established by transwell in vitro, which further demonstrated the antioxidant effects of hUC-MSCs spheroids were better than those of hUC-MSCs suspension.
In summary, reasons for the therapeutic advantages of 3D hUC-MSCs spheroids may include: (1) The histocompatibility of MSCs spheroids after implantation in tissues is enhanced, unaffected by MSCs attachment to the substrate (Li et al. 2015); (2) A hypoxic environment is formed inside MSCs spheroids, which stimulates the secretion of vascular endothelial growth factor (VEGF) and other trophic factors (e.g., VEGF, HGF, EGF, etc.) (Miceli et al. 2019, Bartosh et al. 2010) ; (3) 3D spheroids can more effectively replicate the state of MSCs entering tissues in vivo, maximizing their therapeutic effect (Edmondson et al. 2014, Lee et al. 2022).Therefore, orthotopic transplantation of hUC-MSCs spheroids could be a promising clinical strategy for POF treatment.
Currently, studies have demonstrated that hUC-MSCs primarily improve ovarian function in POF through the following mechanisms: (1) hUC-MSCs secrete angiogenic growth factors VEGF, and promote ovarian vascular remodeling, thereby improving the ovarian microenvironment (Zhang et al. 2022).(2) hUC-MSCs reduce oxidative stress in ovarian tissue and provide an excellent environment for follicle development (Deng et al. 2021)

. (3) hUC-MSCs inhibit autophagy of interstitial membrane cells and
Copyright © 2023 Society of Reproduction and Fertility Downloaded from Bioscientifica.com at 07/07/2024 01:13:25AM via Open Access.This work is licensed under a Creative Commons Attribution 4.0 International License http://creativecommons.org/licenses/by/4.0/deed.en_GBrestore ovarian function (Lu et al. 2020).Reactive oxygen species (ROS) are produced in the mitochondrial respiratory chain process, and moderate ROS facilitates the proliferation and differentiation of GCs, whereas excessive ROS triggers apoptosis and excessive autophagy, thereby leading to follicular development disorders (Dai et al. 2023, Zhou et al. 2023, Wu et al. 2023).A study demonstrated that human placentaderived MSCs restore ovary function through an antioxidant effect (Seok et al. 2020).
Similarly, 3D-cultured placenta-derived MSCs spheroids could enhance ovary function by inducing folliculogenesis (Kim et al. 2018).In addition, a study showed the exosomes from menstrual blood-derived MSCs improved ovarian function by inhibiting follicle apoptosis and increasing GC proliferation, similar to our findings, MSCs can inhibit apoptosis and increase the proliferation of GCs (Zhang et al. 2021).
Additionally, a study showed that heme oxygenase-1 (HO-1) expressed in MSCs could restore the ovarian function of the POF mice model by regulating autophagy and apoptosis of GCs (Yin et al. 2020).These findings support our conclusion that 3D hUC-MSCs spheroids could reduce apoptosis and excessive autophagy of GCs in improving POF.
However, there are still some limitations to this study: (1) Although orthotopic transplantation can increase the planting of hUC-MSCs spheroids in ovarian tissues, the damage caused by surgery may influence the evaluation of the effect of hUC-MSCs on improving POF, therefore optimizing the orthotopic transplantation method remains to be investigated.(2) Although the anti-oxidation, anti-apoptosis, and anti-autophagy effects of 3D hUC-MSCs spheroids were better than those of monolayer cultured hUC- Page 12 of 42 Accepted Manuscript published as REP-23-0496.R2.Accepted for publication: 24-Jun-2024 Copyright © 2023 Society of Reproduction and Fertility Downloaded from Bioscientifica.com at 07/07/2024 01:13:25AM via Open Access.This work is licensed under a Creative Commons Attribution 4.0 International License http://creativecommons.org/licenses/by/4.0/deed.en_GB Figure legends:

Fig. 4 .
Fig. 4. The immunohistochemistry images show the expression of autophagy-related