Therapeutic Potential of Super Activated Platelet Lysate (sPL) and Umbilical Cord Mesenchymal Stem Cells (UCMSCs) in Enhancing Endometrial Regeneration in Rats with Thin Endometrium

Therapeutic Potential of Super Activated Platelet Lysate (sPL) and Umbilical Cord Mesenchymal Stem Cells (UCMSCs) in Enhancing Endometrial Regeneration in Rats with Thin Endometrium | 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 Article Therapeutic Potential of Super Activated Platelet Lysate (sPL) and Umbilical Cord Mesenchymal Stem Cells (UCMSCs) in Enhancing Endometrial Regeneration in Rats with Thin Endometrium Ling-Qi Meng, Yi Zhang, Chun-Xiang Liu, Ihsan Ullah, Tian-Qi Zhang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5870571/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Background This study aimed to establish a rat model of thin endometrium and investigate the effects of super-activated platelet lysate (sPL) and umbilical cord mesenchymal stem cells (UCMSCs) on the thin endometrium in rats. Methods Thin endometrium models were induced by infusing absolute ethyl alcohol into the uteri of female Sprague-Dawley (SD) rats. Rats were randomly assigned to several groups (Normal, Model, Extracellular matrix (ECM) + sPL, ECM + cell, Gel + sPL, Gel + cell) and treated for 21 or 42 days. Histopathological structures and endometrial thickness were observed using hematoxylin-eosin (HE) staining. ELISA was used to detect PDGF-BB, TGF-β1, E2 and FSH expression levels in serum. Furthermore, Western blot and immunohistochemical staining were used to assess the expression levels of cyclin D1, CD34, pan-keratin, cytokeratin 18, and vimentin in uterine tissue. Results HE staining revealed improvements in endometrial thickness, gland number, and blood vessels following treatment with sPL and UCMSCs in the thin endometrium rat model. Compared to the model group, ELISA results demonstrated that the PDGF-BB, E2, TGF-β1 and FSH serum in treatment groups returned to normal levels. Immunohistochemical staining and Western blot results indicated decreased keratin, cytokeratin, and vimentin expression levels in the model group, which were significantly increased by sPL perfusion or UCMSCs transplantation. Conclusion Intrauterine perfusion of sPL improves endometrium thickness, morphology, function, and repair capacity in rats with thin endometrium. The therapeutic efficacy of uterine infusion of sPL surpasses that of UCMSCs transplantation. Biological sciences/Stem cells/Mesenchymal stem cells Health sciences/Diseases/Reproductive disorders/Infertility Thin endometrium Super activated platelet lysate (sPL) Umbilical cord mesenchymal stem cells (UCMSCs) Endometrium regeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Fertility issues have garnered considerable attention within the field of gynecology and obstetrics, with their manifestation suffering adverse influence by both age and various gynecological diseases [ 1 ]. Among the challenges encountered is the presence of a thin endometrium, often accompanied by compromised endometrial receptivity, recognized as a pivotal factor in the success of embryo implantation and prevention of pregnancy failure [ 2 ]. Numerous research has been published in the literature investigating the impact of various therapeutic interventions, particularly biological therapies. Preclinical experiments have demonstrated a significant enhancement in thin endometrium thickness following the infusion of stem cells into the uterus [ 3 ]. Clinical reports further substantiate these findings, revealing a substantial increase in endometrial thickness in patients with thin endometrium following stem cell treatments [ 4 ]. Consequently, estrogen and stem cell therapy emerge as promising modalities for addressing thin endometrium and, thereby, augmenting the likelihood of successful pregnancies [ 5 ]. Previous research has explored the administration of high doses of estrogen, specifically E2, and the utilization of stem cells and amnion to improve thin endometrium thickness [ 6 ]. However, despite these endeavors, clinical outcomes have not significantly improved with the currently available treatments [ 7 ]. Given the pivotal roles that good endometrial receptivity and adequate endometrial thickness play in embryo implantation and subsequent pregnancy [ 8 ], developing effective treatment protocols for patients with thin endometrium remains a substantial challenge requiring prompt solutions. In recent years, Mesenchymal Stem Cell (MSC) therapy has emerged as a novel therapeutic modality for fostering tissue regeneration, leveraging its inherent self-renewal, robust proliferation, and differentiation capacities [ 9 ]. Current investigations underscore the widespread utilization of MSCs in preclinical and clinical settings, encompassing distinct types such as bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, and umbilical cord blood mesenchymal stem cells [ 10 ]. Particular attention has been directed towards Umbilical Cord Mesenchymal Stem Cells (UCMSCs) among the various MSC types due to their facile isolation and versatile application [ 11 ]. Extensive research has revealed that MSCs, including UCMSCs, play a pivotal role in tissue regeneration when employed as standalone cell therapy or in conjunction with biomaterials within the framework of tissue engineering techniques [ 12 ]. The principal functions of MSCs are primarily ascribed to their capabilities in migration and chemotaxis, facilitating subsequent differentiation into functional cells at sites of tissue damage [ 13 ]. Notably, UCMSCs have demonstrated efficacy in promoting tissue regeneration and repairing damaged endometrium in various preclinical applications to address thin endometrium-related therapeutic challenges [ 14 ]. Nevertheless, emerging studies have progressively indicated that transplanted MSCs exhibit limited survival durations, impeding their capacity to fully differentiate into functional cells and contribute significantly to endometrial regeneration. During the reproductive phase in females, the endometrium undergoes a cyclical process of shedding, proliferation, differentiation, and repair, collectively known as menstruation [ 15 ]. The regulatory actions of the steroid hormone estrogen within the endometrial tissue primarily orchestrate this intricate cycle [ 16 ]. Estrogen play pivotal roles in the complex regulation of female reproductive functions, with a significant focus on the uterus and ovaries [ 17 ]. The management of uterine growth, contraction, endometrial physiology, menstruation, and the maintenance of a successful pregnancy has traditionally centered around controlling estrogen and progesterone (Pg) levels through hormonal therapies [ 18 ]. Super-active platelet lysate (sPL) is a rapid release of high-concentration bioactive factors in platelets based on the patented platelet-efficient induction–activation culture technique. An emerging approach involves using super-activated platelet lysate (sPL) derived from platelet-rich plasma (PRP) through ultra-low temperature freeze-thawing. sPL exhibits elevated levels of growth factors, including platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF), etc [ 19 ]. These growth factors promote tissue regeneration and inhibit local inflammatory responses [ 20 ]. SPL has higher safety than PRP and can be stored for a longer period of time. During preparation of sPL, residual solid cellular components and white cells are removed, while immunogenicity is reduced. sPL can be directly injected into the damage position without activating platelets. Compared to traditional PRP, sPL can enhance hormone secretion, directly activate MSCs and expedite tissue regeneration [ 21 ]. Furthermore, there is a need to develop more specific protocols for applying sPL in gynecological diseases. Additionally, further exploration warrants the treatment of hormone-dependent and immune-related disorders using sPL. We established a thin endometrium model to assess the potential pharmacological application of sPL in a clinical context. Subsequently, we performed comprehensive histological, biological, and functional analyses to explore the potential of sPL or UCMSCs administration in reinstating endometrial functionality and enhancing pregnancy outcomes. 2. Results 2.1 Effect of sPL and UCMSCs on the morphology of endometrium H&E staining showed that the uterine tissues observed visible blood vessels and glands, and the cells were tightly arranged in the endometrium of rats. The uterus in the model group was significantly thin and had extensive necrosis of the endometrium in nearly the entire layer of the endometrium. Rats in the sPL and UCMSCs treatment groups had significantly thicker endometrium and increased endometrial glands compared with the model group after 21 days of treatment (Fig. 1 ). The structure of epithelium was more integrity in treatment groups. The endometrial thicknesses of the Normal, Model 21, ECM + sPL 21, ECM + cell 21, Gel + sPL 21, and Gel + cell 21 was showed as follows: 917.5 ± 83.6 µm, 181.1 ± 41.2 µm, 538.5 ± 51.6 µm, 360.6 ± 63.7 µm, 546.8 ± 73.2 µm and 339.8 ± 74.9 µm, respectively (Table 1 ). The treatment groups of sPL and UCMSCs showed relatively intact endometrium structures, with increased capillaries and glands and thicker endometrium at 42 days. There was no significant difference between the treatment and the normal groups after 42 days of treatment (Fig. 1 B and C). In 21 days and 42 days treatment group, the number of endometrial glands was increased significantly compared with the Model group (Fig. 1 D and E). The effects of 21 Days groups and 42 Days groups remodeling uterine structure, endometrial glandsand epithelial repair remained stable. Table 1 Thickness of the endometrium in the different groups Group Thickness of endometrium (µm) Weight (g) Normal 917.5 ± 83.6 346.5 ± 5.3 Model 21 181.1 ± 41.2 345.2 ± 4.5 Model 42 462.7 ± 66.4 351.3 ± 6.1 ECM + sPL 21 538.5 ± 51.6** 328.6 ± 9.7 ECM + cell 21 360.6 ± 63.7* 350.7 ± 7.1 Gel + sPL 21 546.8 ± 73.2** 369.0 ± 5.6 Gel + cell 21 339.8 ± 74.9* 369.9 ± 10.2 ECM + sPL 42 742.8 ± 85.2† 392.8 ± 12.1 ECM + cell 42 541.7 ± 82.0 389.6 ± 8.2 Gel + sPL 42 682.6 ± 54.8 381.6 ± 9.6 Gel + cell 42 786.3 ± 71.5† 382.5 ± 9.9 Compared with the Model 21 group, * P < 0.05, ** P < 0.01. Compared with the Model 42 group, † P < 0.05. n = 6. 2.2 Protein contents in endometrium after sPL and UCMSCs treatment The expression of cyclin D1, CD34, and cytokeratin was examined using IHC to assess the effect of sPL and UCMSCs transplantation on a thin endometrium. According to IHC staining, cyclin D1 and cytokeratin were predominantly expressed in the cytoplasm of endometrial epithelial cells, and CD34 and vimentin in the cytoplasm of endometrial stroma cells (Fig. 2 ). Based on the results, we found that the fluorescence intensities of cyclin D1, CD34, vimentin, and cytokeratin in the normal, sPL, and UCMSCs groups were significantly higher than in the model group ( P < 0.01). Western blot results showed that sPL and UCMSCs increased CK18 and stromal cell marker vimentin expression levels in a thin-endometrium rat model. Cyclin protein changes are a sign of cell proliferation. In comparison with the model group, cyclin D1 and CD34 expression were higher in the treatment group, but no significant differences were found between sPL and UCMSC treatments (Figs. 2 and 3 ). A higher level of cytokeratin and vimentin expression was observed in the sPL group ( P < 0.01) compared to the blank normal group ( P < 0.01). There were no statistically significant differences between the normal and sPL groups. A significant difference was observed in the expression of cytokeratin and vimentin between the subcutaneous and model groups (Figs. 2 and 3 ). Based on Western blot analysis, the experimental group showed significantly higher cytokeratin and vimentin expression than the normal group ( P < 0.05). In contrast, no significant difference was observed between the normal and experimental groups (Fig. 3 ). 2.3 sPL and UCMSCs promote the expression levels of PDGF-BB, E2, TGF-β1 and FSH Compared with the normal group, serum PDGF-BB and TGF-β1 levels in the model groups were increased ( P < 0.05), E2 and FSH levels were decreased ( P < 0.05). After 21 days or 42 days of treatment, compared with the model group, the levels of the PDGF-BB, TGF-β1, E2 and FSH in the sPL group and UCMSCs group were decreased ( P < 0.05). Based on (Fig. 4 ), there was no statistical difference between the two groups. 3. Discussion It has been estimated that the endometrium undergoes more than 400 cycles of development, growth, and shed during a woman's reproductive years. Endometrial thinness occurs as a result of inflammation, endometrial dysfunction, and uterine surgery. To gain a better understanding of the mechanisms underlying endometrial injury and repair, this study investigated the effects of sPL and UCMSCs on thin endometrium. Using histomorphology, Western blotting, and immunocytochemistry to diagnose and repair the damaged microenvironment. It is well-established that absolute ethyl alcohol perfusion in rats induces endometrial injury, Offering advantages such as easy modeling and high success rates. Several in vivo experiments demonstrated that the morphology and thickness of the endometrium were both significantly improved by sPL and UCMSCs. In previous studies, UCMSCs have been shown to thicken the endometrium, increase glands, and increase sub-endometrial angiogenesis, all of which improve pregnancy rates [ 22 ]. The therapeutic effectiveness of sPL significantly exceeded that of UCMSC transplantation in rats with thin endometrium by enhancing endometrial thickness, morphology, function, and repair capacity. Our study suggests that sPL combined with UCMSC therapy may be a promising approach for treating thin endometrium. This study suggests that intrauterine administration of autologous PRP has proliferative and anti-fibrotic effects on damaged endometrium. PRP contains growth factors and cytokines contributing to rapid healing and tissue regeneration by accelerating cell proliferation, angiogenesis, and migration [ 23 ]. However, PRP has not been studied specifically on the endometrium. However, its major growth factor, PDGF, has been demonstrated to play several pivotal roles in endometrial cell proliferation [ 24 ]. Diverse growth factors such as EGF, FGF, TGF-β, insulin-like growth factors, and colony-stimulating factors, along with their corresponding receptors, have been recognized for their participation in autocrine and paracrine functions, influencing endometrial cell growth, differentiation, and implantation [ 25 – 27 ]. Moreover, to the extent of our knowledge, this study is the first to reveal that autologous sPL can enhance endometrial proliferation, decrease fibrosis, and mitigate intrauterine adhesions by using a thin endometrial model. In a recent study, SPL has been demonstrated to have an anti-fibrotic effect on skeletal muscle healing after injury [ 28 ]. However, the result of this study showed that sPL inhibited excessive collagen deposition, which may decrease fibrosis progression in thin endometriums. Compared with the model group, the thickness of the endometrium and the number of glands in sPL or UCMSC treatment groups were significantly higher, and the interstitial blood vessels were denser. A significant increase in endometrial thickness and gland number was observed in the sPL-treated or UCMSC-treated groups, and interstitial blood vessels were decreased significantly. Vimentin and cytokeratin are often considered specific epithelial cell markers [ 29 ]. In endometrial cells, keratin and vimentin are important components of the intermediate filament protein cytoskeleton [ 30 ]. Their role is crucial in promoting cell proliferation and differentiation, and they are often considered dynamic structures [ 29 , 31 ]. In rat endometrial epithelial cells and luminal epithelial cells, cytokeratin 18 is predominantly found in the cytoplasm [ 32 ]. Within the mesenchyme cytoplasm of rats, vimentin is predominantly expressed in stromal cells [ 33 ]. Rat endometrium was found to have high levels of CK18 and vimentin after intrauterine perfusion with sPL and UCMSC. Microscopic examination reveals an intact and tight arrangement of the glandular epithelium and stroma of the endometrium due to changes in endometrial thickness and cyclin D1, CD34, CK18, and vimentin expression levels. Our research showed that sPL and UCMSC perfusion could restore endometrial thickness after intrauterine perfusion and facilitate the rejuvenation of glandular epithelial cells and stromal cells in a thin endometrial structure. According to previous studies, estrogen and progesterone regulate VEGF expression in the endometrium, transforming it from thin and dense to thick and transparent [ 34 ]. VEGF could influence the endometrium's proliferative, secretory, and shedding in combination with female hormones [ 35 ]. Low levels of VEGF in the menstrual phase are associated with endometrial shedding [ 36 ]. In contrast, high levels of VEGF in the secretory phase promote neovascularization and vascular permeability, both of which are beneficial to endometrium repair and embryo implantation during pregnancy [ 37 ]. In our study, VEGF was expressed throughout the implantation window in rat endometrium, suggesting that it could contribute to endometrial receptivity. Furthermore, SPL has also been applied to medical and surgical fields, including skin rejuvenation and hair regrowth, lung dysfunction, neural repair, and orthopedics due to its no-side effects properties. In conclusion, sPL and MSCs can significantly promote endometrium growth, offers significant benefits for thin endometrium treatments, providing a therapeutic option for thin endometrium. Despite ongoing challenges and difficulties, sPL shows significant promise for future clinical trials and could provide an alternative to PRP-based treatments. Furthermore, these promising results may contribute to the development of innovative therapeutic strategies for conditions associated with endometrial dysfunction. The combination of sPL and UCMSCs emerges as a potential candidate for clinical interventions aimed at enhancing endometrial regeneration and improving reproductive outcomes. sPL and MSCs hold great translational potential for regenerative medicine. We believe that further studies are necessary to determine the composite sPL + MSCs was generally superior to its corresponding monotherapy. 4. Materials and Methods 4.1 Animal Study 4.1.1 Animals’ experiments Specific pathogen-free Sprague-Dawley females’ rats (aged 8–10 weeks, weighing 220 ± 20 g) were purchased from Heilongjiang University of Chinese Medicine. All the Rats were housed in a specific virus-free laboratory at an ambient temperature of 23°C, and a 12-hour light/dark cycle and ad libitum access to food and water were provided after one week of acclimatization. The experiment was conducted on rats with complete sexual cycles. 4.1.2 sPL preparations An average of 8 mL of blood was extracted from 20 anaesthetized rats by cardiac puncture and collected in 15 mL centrifuge tube containing 20 U sodium heparin. The red blood cells were removed, and the platelet was collected by centrifuging fresh blood at 1,500 g for 15 min. Platelet activation was achieved by incubating PRP with 20 mM CaCl 2 and 2 U/mL sodium heparin for 2 h at 37°C. Then, the mixture was frozen at -80°C for 2 h, followed by thawing at 37°C and cooling at 4°C to allow fibrin coalescence. sPL is collected from the platelet through ultra-low temperature freeze-thawing. After centrifugation at 3,000 g for 8 min, the supernatant was filtered through a 0.22 µm filter (Millipore, Darmstadt, Germany) to obtain the sPL. A total of 16 mL sPL was collected from 160 mL blood, the prepared and actived sPL was frozen and stored at -20°C. 4.2 Culture and amplification of UCMSCs The UCMSCs were from the mothers who just gave birth, and informed consent was obtained from hospitalized mothers at The First Affiliated Hospital of Harbin Medical University, and rapidly processed. The Ethics Committee of Heilongjiang University of Chinese Medicine approved this study. All experiments were performed in accordance with relevant guidelines and regulations. The cells were harvested and cultured by centrifugation at 1,200 g for 10 min. The cells were resuspended and cultured in DMEM/F12 media with 10% fetal calf serum and 1% P/S at 37°C in a humidified incubator with 5% CO 2 . Three times a week, the culture medium was changed. 0.05% trypsin-EDTA was used to harvest adherent UCMSCs at 90% confluence. After obtaining the third passage of culture, the UCMSCs were used for transplantation. Trypsin-EDTA was used to harvest the cells, followed by two washes with DMEM/F12 before resuspending them at 62,500 cells/µL in DMEM/F12. 4.3 Preparation of injection mixtures To obtain a proper mixture, an efficient dispersion of the composition of the mixture must be provided to avoid precipitation. The chitosan powders were dissolved in 0.01 mol/L of acetic acid solution. The mixture was heated to 60°C under continuous magnetic stirring (200 rpm) for 180 min for better dissolution and cooling to 4°C. This can be accomplished through a three-way stopcock device, connecting a syringe with 160 µL sPL or UCMSCs on one side and another syringe with 40µL 5% chitosan (Sigma-Aldrich, St. Louis, USA) or extracellular matrix (ECM, 354263, BD Biosciences, Bedford MA, USA) on the opposite side. The device ensures a homogeneous mixture, consolidating components within a single syringe for injection. 4.4 Model preparation and treatment The estrus status of rats was monitored daily through vaginal smears. Nembutal sodium was used for general anesthesia during the operation on rats in stable estrus. Rats were placed in the supine position on the operating table, and the lower abdomen was scraped and disinfected with ethanol. The abdomen skin and muscles were deeply opened, starting with a longitudinal incision of 1.5 to 2 cm, followed by layer-by-layer openings. Near the vagina and ovary, the uterus was ligated. Then, 200–300 µL PBS was kept in the uterus for 5 min in the normal groups and 200–300 µL 95% ethanol was injected in the model and treatment groups for 5 min, respectively. In addition to extracting the remaining ethanol, the uterine cavities were washed twice with physiological saline. Three rounds of iodophor were applied to the incision site and sutured to return the uterus to its normal anatomical position after the incision was made. The normal group was maintained normally without intervention. Model rats were divided into five groups: Model group (40 µL 5% chitosan, 40 µL ECM and 120 µL PBS / rat, n = 20), ECM + sPL group (40 µL ECM and 160 µL sPL / rat, n = 20), ECM + cell group (40 µL ECM and 1 × 10 7 UCMSC/160 µL / rat, n = 20), Gel + sPL group (40 µL 5% chitosan and 160 µL sPL/ rat, n = 20), and Gel + cell group (40 µL 5% chitosan and 1 × 10 7 UCMSC/160 µL / rat, n = 20). After modeling for 6 to 8 h, we perfused the therapeutic mixture into the uterus cavity with a medical syringe. To evaluate the endometrium status, rats were anesthetized for sampling at 21 days and 42 days after treatment. To further investigate, the uterus of rats was sectioned and preserved at -80°C or 4% paraformaldehyde. 4.5 Histopathological examinations The rat uterus was removed in each group and placed in paraffin. The uterus was sliced at a thickness of 5 µm and dried at 45°C. The sections were covered in xylene and ethanol, varying from high to low concentrations. We stained the sections with HE stain kit (G1120, Solarbio), and used CaseViewer image processing software (3DHISTECH, Hungary) to analyze the vertical distance between the uterine cavity and the myometrium. A comparison of group differences in endometrial morphology and thickness was made by obtaining data from five random views of the endometrium. 4.6 Immunohistochemistry The uterine was stored in 4% paraformaldehyde and placed in wax blocks. To block endogenous peroxidase activity and non-specific antigens, the sections were deparaffinized in xylene, rehydrated with ethanol, then incubated with 3% H 2 O 2 for 15 min and 10% goat serum for 20 min. The sections were incubated with the primary antibodies CD34 (1: 2,500, ab81289, Abcam), cyclin D1 (1: 100, ab134175, Abcam), cytokeratin 18 (1: 500, ab133263, Abcam) and vimentin (1: 200, sc-6260, Santa Cruz) at a dilution of 1: 100 overnight at 4°C. Slides were incubated with 50 µL secondary antibodies and followed by 3,3′-diaminobenzidine tetrahydrochloride (DAB) solution for 1 h. A hematoxylin solution was then applied to the slides for 3 min, and the slides were analyzed with CaseViewer image processing software (3DHISTECH, Hungary). 4.7 Western Blot analysis Uterine tissues were lysed by RIPA lysis buffer to extract proteins. After lysing, the Uterine tissues in 400 µL lysis buffer (P0013C, Beyotime) and protein (30 µg / 20 µL) were stained with loading dye, separated with 8% − 12% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes and incubated with 5% skim milk. The membranes were exposed to cyclin D1 antibody (1: 2,000, ab134175, Abcam), CD34 antibody (1: 5,000, ab81289, Abcam), Pan-keratin antibody (1: 1,000, #4545, cell signaling technology), cytokeratin 18 antibody (1: 1,000, ab133263, Abcam) and vimentin antibody (1: 200, sc-6260, Santa Cruz) overnight. The membranes were washed with TBST and incubated with secondary anti-rabbit (1: 5,000, ab6721, Abcam) or anti-mouse IgG (1: 5,000, ab6789, Abcam) for 1 h at room temperature. The chemiluminescent signal was developed using the ECL Kit (Invitrogen, Waltham, MA, USA) and the protein contents was detected using ImageJ 1.52a (National Institutes of Health, USA). 4.8 Biochemical and Histopathological Analysis All blood samples collected by abdominal aortic blood were stood at room temperature for 1 h, then the samples were centrifuged at 6,000 g for 15 min to collect the serums. The serums extracted from the blood samples of the experimental rats were separated and stored at -20°C. The concentrations of PDGF-BB, E2, TGF-β1 and FSH were performed using ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA) according to the instructions provided by the manufacturer. 4.9 Ethics statement The animal study was reviewed and approved by the Ethical Committee for Animal Care and Use of the Heilongjiang University of Chinese Medicine (Ethics approval number: 2024011615). All authors confirm that all animal procedures and care were performed in accordance with relevant guidelines and regulations. All animal experiments were performed in accordance with the ARRIVE guidelines. 4.10 Statistical analysis Analysis was performed using the statistical software SPSS 22.0. The data in our study are presented as mean ± standard deviation (SD). Students' T-tests, or a one-way analysis of variance (ANOVA), were used for comparisons between the two groups. Statistical significance was indicated by P < 0.05. Declarations Acknowledgments The authors thank to Ethical Committee for Animal Care and Use of the Heilongjiang University of Chinese Medicine for animal experiment support of this article (Ethics approval number: 2024011615). Author Contributions L.Q. M.†: Conceptualization, Methodology, Software, Validation, Investigation, Data Curation, Writing – Original Draft, Visualization; Y. Z.†: Conceptualization, Methodology, Writing – Original Draft; C.X. L.: Methodology, Validation, Data Curation; I.U.: Writing – Review & Editing; T.Q. Z.: Data Curation; Y. Z.*: Conceptualization, Validation, Investigation, Supervision, writing – review and editing. All authors reviewed the manuscript. Data availability Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the corresponding authors (Ling-Qi Meng: [email protected] and Yi Zhang: [email protected] ) upon reasonable request. Funding No funding was received for conducting this study. Ethics Approval All animal experiments were performed in accordance with the ARRIVE guidelines. Approval was granted by the Ethics Committee of University of Chinese Medicine (Ethics approval number: 2024011615). Competing Interests The authors declare no competing interests. Consent for Publication All authors gave consent for publication. ORCID ID: Ling-Qi Meng†: 0009-0007-8288-5111. Yi Zhang*: 0000-0002-4012-8258. References AAyhan, A. et al. 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Tissue Bank. 24 , 285–306. 10.1007/s10561-022-10039-z (2023). Zhang, X. et al. PDGFBB improved the biological function of menstrual blood-derived stromal cells and the anti-fibrotic properties of exosomes. Stem Cell. Res. Ther. 14 , 113. 10.1186/s13287-023-03339-y (2023). Apte, R. S., Chen, D. S. & Ferrara, N. VEGF in Signaling and Disease: Beyond Discovery and Development. Cell 176 , 1248–1264. 10.1016/j.cell.2019.01.021 (2019). Hajipour, H. et al. An update on platelet-rich plasma (PRP) therapy in endometrium and ovary related infertilities: clinical and molecular aspects. Syst. Biol. Reprod. Med. 67 , 177–188. 10.1080/19396368.2020.1862357 (2021). Ma, J. et al. Recent trends in therapeutic strategies for repairing endometrial tissue in intrauterine adhesion. Biomater. Res. 25 , 40. 10.1186/s40824-021-00242-6 (2021). Guo, X. et al. Effect of super activated platelet lysate on cell proliferation, repair and osteogenesis. Biomed. Mater. Eng. 34 , 95–109. 10.3233/BME-221426 (2023). Wang, T. & Tan, J. Therapeutic effect of menstrual blood stem cells in rats with thin endometrium. Zhejiang Da Xue Xue Bao Yi Xue Ban . 52 , 13–23. 10.3724/zdxbyxb-2022-0509 (2023). Chenglin Miao, S., Zhao, S. E. M. & Yaming Jiu. The diverse actions of cytoskeletal vimentin in bacterial infection and host defense. J. Cell. Sci. 136 , jcs260509. 10.1242/jcs.260509 (2023). Nick, A., Kuburich, P., den Hollander, J. T., Pietz, Sendurai, A. & Mani Vimentin and cytokeratin: Good alone, bad together. Semin Cancer Biol. 86 , 816–826. 10.1016/j.semcancer.2021.12.006 (2022). Tantengco, O. A. G. et al. Progesterone alters human cervical epithelial and stromal cell transition and migration: Implications in cervical remodeling during pregnancy and parturition. Mol. Cell. Endocrinol. 529 , 111276. 10.1016/j.mce.2021.111276 (2021). Chen, K. et al. A Novel Method to Repair Thin Endometrium and Restore Fertility Based on Menstruation-Derived Stem Cell. Reprod. Sci. 31 , 1662–1673. 10.1007/s43032-024-01458-2 (2024). Jianghong et al. Progress on the Role of Estrogen and Progesterone Signaling in Mouse Embryo Implantation and Decidualization. Reproductive Sci. 30 , 1746–1757. 10.1007/s43032-023-01169-0 (2023). Hu, X. et al. Cyclical endometrial repair and regeneration: Molecular mechanisms, diseases, and therapeutic interventions. MedComm 4, e425. (2020). 10.1002/mco2.425 (2023). Naseri, S. et al. A cross-sectional study comparing the inflammatory profile of menstrual effluent vs. peripheral blood. Health Sci. Rep. 6 , e1038. 10.1002/hsr2.1038 (2023). Pérez-Gutiérrez, L. & Ferrara, N. Biology and therapeutic targeting of vascular endothelial growth factor A. Nat. Rev. Mol. Cell Biol. 24 , 816–834. 10.1038/s41580-023-00631-w (2023). Additional Declarations No competing interests reported. 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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-5870571","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":437843183,"identity":"f1b84393-b6c2-4924-adcc-de6f830f4614","order_by":0,"name":"Ling-Qi Meng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAw0lEQVRIiWNgGAWjYBAC+/b2ww8/VNjUN7Y3EKnFgOdMmrHEmTTG5p4DxGqRSDCQ4G05xNg+I4FILeYMCQkGkg0HmHlnPt54g6HGJpqgFsuGgwceFO64wyY5O63YguFYWm4DQT0HG4C2nHnGYzg7x0yCseEwEVoOA73D23ZYwv7mGSK1GByDaDFgnMFDpBbJHh5wICcw9gD9kkCMX/jln4OjMoGx/fDGGx9qbIjwC7IjJRJIUQ7RQqqOUTAKRsEoGBkAAGOjRQoUaVaEAAAAAElFTkSuQmCC","orcid":"","institution":"Tian Qing Stem Cell Co. Ltd","correspondingAuthor":true,"prefix":"","firstName":"Ling-Qi","middleName":"","lastName":"Meng","suffix":""},{"id":437843184,"identity":"167966ef-a0d2-451f-b884-eba00a6ad10c","order_by":1,"name":"Yi Zhang","email":"","orcid":"","institution":"Heilongjiang University of Chinese Medicine), Ministry of Education","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Zhang","suffix":""},{"id":437843185,"identity":"068a9d52-4570-4bec-bbe5-e4e6ce4c1817","order_by":2,"name":"Chun-Xiang Liu","email":"","orcid":"","institution":"Tian Qing Stem Cell Co. Ltd","correspondingAuthor":false,"prefix":"","firstName":"Chun-Xiang","middleName":"","lastName":"Liu","suffix":""},{"id":437843187,"identity":"61bd6600-8cbf-4b2a-81aa-1f363d19e46c","order_by":3,"name":"Ihsan Ullah","email":"","orcid":"","institution":"Tian Qing Stem Cell Co. Ltd","correspondingAuthor":false,"prefix":"","firstName":"Ihsan","middleName":"","lastName":"Ullah","suffix":""},{"id":437843189,"identity":"9a4c7259-9854-4b27-a121-b8393fc223bd","order_by":4,"name":"Tian-Qi Zhang","email":"","orcid":"","institution":"Tian Qing Stem Cell Co. Ltd","correspondingAuthor":false,"prefix":"","firstName":"Tian-Qi","middleName":"","lastName":"Zhang","suffix":""},{"id":437843190,"identity":"f88664e0-68a0-410c-b209-21178c3b2eea","order_by":5,"name":"Yi Zhang","email":"","orcid":"","institution":"Tian Qing Stem Cell Co. Ltd","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-01-21 06:23:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5870571/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5870571/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-10587-w","type":"published","date":"2025-07-09T15:58:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79885107,"identity":"77c8e411-a4d7-44d0-9c72-f6090bbbcc0e","added_by":"auto","created_at":"2025-04-04 05:40:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1424213,"visible":true,"origin":"","legend":"\u003cp\u003eHE staining shows the morphology of the endometrium. Results of HE analysis of each group (scale bar = 1,000 μm); (B) Results of endometrial thicknesses after 21 days treatment, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; (C) Results of endometrial thicknesses after 42 days treatment, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05. **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; (D) Results of Glands number after 21 days treatment, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001; (E) Results of Glands number after 42 days treatment, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001. n = 6.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5870571/v1/43495fc52f5360ef217ac5e3.png"},{"id":79885112,"identity":"0c46a149-2208-4b3e-8e89-f8aedf749538","added_by":"auto","created_at":"2025-04-04 05:40:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2593949,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of sPL and UCMSCs transplantation on endometrial epithelial with immunohistochemistry. The results of cyclin D1, CD34, cytokeratin and vimentin (up: × 100 and down: × 400, scale bar = 100 μm ). n = 6.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5870571/v1/f7c7e9f5adf7de6c5aa3b4fe.png"},{"id":79886432,"identity":"31f26a08-ebe2-4317-ba11-f02ccb04a289","added_by":"auto","created_at":"2025-04-04 06:05:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":660131,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of sPL and UCMSCs transplantation of the endometrium. (A) The expression of the cyclin D1, CD34, keratin, cytokeratin 18 and vimentin protein in each group was detected by western blotting. The original images are shown in the Appendix. (B) A histogram shows the relative expression level of the target proteins to that of an internal standard. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. n = 3 uteri.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5870571/v1/29485a0df8b3c7449bbbbae5.png"},{"id":79885108,"identity":"c0496884-84cb-4ba1-bb70-3ddf20219629","added_by":"auto","created_at":"2025-04-04 05:40:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":746622,"visible":true,"origin":"","legend":"\u003cp\u003eSerum levels of PDGF-BB, E2, TGF-β1 and FSH in the rat model of thin endometrium. Expression of (A) PDGF-BB, (B) E2, (C) TGF-β1, (D) FSH in the serum of rats. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01 vs. model. n = 6.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5870571/v1/8f8be9003f650c24fb910763.png"},{"id":86699567,"identity":"baea1696-da86-40dc-9394-6b9352d8419c","added_by":"auto","created_at":"2025-07-14 16:11:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6026358,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5870571/v1/e2ba0f5a-8dc8-4c9e-a014-07c094799c48.pdf"},{"id":79886372,"identity":"682d53f4-7680-4161-b489-3115f831d9a1","added_by":"auto","created_at":"2025-04-04 06:05:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":144632,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5870571/v1/2a386eca84eea817656d2dc3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Therapeutic Potential of Super Activated Platelet Lysate (sPL) and Umbilical Cord Mesenchymal Stem Cells (UCMSCs) in Enhancing Endometrial Regeneration in Rats with Thin Endometrium","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eFertility issues have garnered considerable attention within the field of gynecology and obstetrics, with their manifestation suffering adverse influence by both age and various gynecological diseases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Among the challenges encountered is the presence of a thin endometrium, often accompanied by compromised endometrial receptivity, recognized as a pivotal factor in the success of embryo implantation and prevention of pregnancy failure [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Numerous research has been published in the literature investigating the impact of various therapeutic interventions, particularly biological therapies. Preclinical experiments have demonstrated a significant enhancement in thin endometrium thickness following the infusion of stem cells into the uterus [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Clinical reports further substantiate these findings, revealing a substantial increase in endometrial thickness in patients with thin endometrium following stem cell treatments [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConsequently, estrogen and stem cell therapy emerge as promising modalities for addressing thin endometrium and, thereby, augmenting the likelihood of successful pregnancies [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Previous research has explored the administration of high doses of estrogen, specifically E2, and the utilization of stem cells and amnion to improve thin endometrium thickness [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, despite these endeavors, clinical outcomes have not significantly improved with the currently available treatments [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Given the pivotal roles that good endometrial receptivity and adequate endometrial thickness play in embryo implantation and subsequent pregnancy [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], developing effective treatment protocols for patients with thin endometrium remains a substantial challenge requiring prompt solutions.\u003c/p\u003e \u003cp\u003eIn recent years, Mesenchymal Stem Cell (MSC) therapy has emerged as a novel therapeutic modality for fostering tissue regeneration, leveraging its inherent self-renewal, robust proliferation, and differentiation capacities [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Current investigations underscore the widespread utilization of MSCs in preclinical and clinical settings, encompassing distinct types such as bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, and umbilical cord blood mesenchymal stem cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Particular attention has been directed towards Umbilical Cord Mesenchymal Stem Cells (UCMSCs) among the various MSC types due to their facile isolation and versatile application [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Extensive research has revealed that MSCs, including UCMSCs, play a pivotal role in tissue regeneration when employed as standalone cell therapy or in conjunction with biomaterials within the framework of tissue engineering techniques [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The principal functions of MSCs are primarily ascribed to their capabilities in migration and chemotaxis, facilitating subsequent differentiation into functional cells at sites of tissue damage [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Notably, UCMSCs have demonstrated efficacy in promoting tissue regeneration and repairing damaged endometrium in various preclinical applications to address thin endometrium-related therapeutic challenges [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNevertheless, emerging studies have progressively indicated that transplanted MSCs exhibit limited survival durations, impeding their capacity to fully differentiate into functional cells and contribute significantly to endometrial regeneration. During the reproductive phase in females, the endometrium undergoes a cyclical process of shedding, proliferation, differentiation, and repair, collectively known as menstruation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The regulatory actions of the steroid hormone estrogen within the endometrial tissue primarily orchestrate this intricate cycle [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Estrogen play pivotal roles in the complex regulation of female reproductive functions, with a significant focus on the uterus and ovaries [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The management of uterine growth, contraction, endometrial physiology, menstruation, and the maintenance of a successful pregnancy has traditionally centered around controlling estrogen and progesterone (Pg) levels through hormonal therapies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Super-active platelet lysate (sPL) is a rapid release of high-concentration bioactive factors in platelets based on the patented platelet-efficient induction\u0026ndash;activation culture technique. An emerging approach involves using super-activated platelet lysate (sPL) derived from platelet-rich plasma (PRP) through ultra-low temperature freeze-thawing. sPL exhibits elevated levels of growth factors, including platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF), etc [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These growth factors promote tissue regeneration and inhibit local inflammatory responses [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. SPL has higher safety than PRP and can be stored for a longer period of time. During preparation of sPL, residual solid cellular components and white cells are removed, while immunogenicity is reduced. sPL can be directly injected into the damage position without activating platelets. Compared to traditional PRP, sPL can enhance hormone secretion, directly activate MSCs and expedite tissue regeneration [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Furthermore, there is a need to develop more specific protocols for applying sPL in gynecological diseases. Additionally, further exploration warrants the treatment of hormone-dependent and immune-related disorders using sPL.\u003c/p\u003e \u003cp\u003eWe established a thin endometrium model to assess the potential pharmacological application of sPL in a clinical context. Subsequently, we performed comprehensive histological, biological, and functional analyses to explore the potential of sPL or UCMSCs administration in reinstating endometrial functionality and enhancing pregnancy outcomes.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Effect of sPL and UCMSCs on the morphology of endometrium\u003c/h2\u003e \u003cp\u003eH\u0026amp;E staining showed that the uterine tissues observed visible blood vessels and glands, and the cells were tightly arranged in the endometrium of rats. The uterus in the model group was significantly thin and had extensive necrosis of the endometrium in nearly the entire layer of the endometrium. Rats in the sPL and UCMSCs treatment groups had significantly thicker endometrium and increased endometrial glands compared with the model group after 21 days of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The structure of epithelium was more integrity in treatment groups. The endometrial thicknesses of the Normal, Model 21, ECM\u0026thinsp;+\u0026thinsp;sPL 21, ECM\u0026thinsp;+\u0026thinsp;cell 21, Gel\u0026thinsp;+\u0026thinsp;sPL 21, and Gel\u0026thinsp;+\u0026thinsp;cell 21 was showed as follows: 917.5\u0026thinsp;\u0026plusmn;\u0026thinsp;83.6 \u0026micro;m, 181.1\u0026thinsp;\u0026plusmn;\u0026thinsp;41.2 \u0026micro;m, 538.5\u0026thinsp;\u0026plusmn;\u0026thinsp;51.6 \u0026micro;m, 360.6\u0026thinsp;\u0026plusmn;\u0026thinsp;63.7 \u0026micro;m, 546.8\u0026thinsp;\u0026plusmn;\u0026thinsp;73.2 \u0026micro;m and 339.8\u0026thinsp;\u0026plusmn;\u0026thinsp;74.9 \u0026micro;m, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The treatment groups of sPL and UCMSCs showed relatively intact endometrium structures, with increased capillaries and glands and thicker endometrium at 42 days. There was no significant difference between the treatment and the normal groups after 42 days of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and C). In 21 days and 42 days treatment group, the number of endometrial glands was increased significantly compared with the Model group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and E). The effects of 21 Days groups and 42 Days groups remodeling uterine structure, endometrial glandsand epithelial repair remained stable.\u003c/p\u003e \u003cp\u003e \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\u003eThickness of the endometrium in the different groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThickness of endometrium (\u0026micro;m)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWeight (g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e917.5\u0026thinsp;\u0026plusmn;\u0026thinsp;83.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e346.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eModel 21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e181.1\u0026thinsp;\u0026plusmn;\u0026thinsp;41.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e345.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eModel 42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e462.7\u0026thinsp;\u0026plusmn;\u0026thinsp;66.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e351.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eECM\u0026thinsp;+\u0026thinsp;sPL 21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e538.5\u0026thinsp;\u0026plusmn;\u0026thinsp;51.6**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e328.6\u0026thinsp;\u0026plusmn;\u0026thinsp;9.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eECM\u0026thinsp;+\u0026thinsp;cell 21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e360.6\u0026thinsp;\u0026plusmn;\u0026thinsp;63.7*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e350.7\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGel\u0026thinsp;+\u0026thinsp;sPL 21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e546.8\u0026thinsp;\u0026plusmn;\u0026thinsp;73.2**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e369.0\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGel\u0026thinsp;+\u0026thinsp;cell 21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e339.8\u0026thinsp;\u0026plusmn;\u0026thinsp;74.9*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e369.9\u0026thinsp;\u0026plusmn;\u0026thinsp;10.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eECM\u0026thinsp;+\u0026thinsp;sPL 42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e742.8\u0026thinsp;\u0026plusmn;\u0026thinsp;85.2\u0026dagger;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e392.8\u0026thinsp;\u0026plusmn;\u0026thinsp;12.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eECM\u0026thinsp;+\u0026thinsp;cell 42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e541.7\u0026thinsp;\u0026plusmn;\u0026thinsp;82.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e389.6\u0026thinsp;\u0026plusmn;\u0026thinsp;8.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGel\u0026thinsp;+\u0026thinsp;sPL 42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e682.6\u0026thinsp;\u0026plusmn;\u0026thinsp;54.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e381.6\u0026thinsp;\u0026plusmn;\u0026thinsp;9.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGel\u0026thinsp;+\u0026thinsp;cell 42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e786.3\u0026thinsp;\u0026plusmn;\u0026thinsp;71.5\u0026dagger;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e382.5\u0026thinsp;\u0026plusmn;\u0026thinsp;9.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eCompared with the Model 21 group, * \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01. Compared with the Model 42 group, \u0026dagger; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. n\u0026thinsp;=\u0026thinsp;6.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Protein contents in endometrium after sPL and UCMSCs treatment\u003c/h2\u003e \u003cp\u003eThe expression of cyclin D1, CD34, and cytokeratin was examined using IHC to assess the effect of sPL and UCMSCs transplantation on a thin endometrium. According to IHC staining, cyclin D1 and cytokeratin were predominantly expressed in the cytoplasm of endometrial epithelial cells, and CD34 and vimentin in the cytoplasm of endometrial stroma cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Based on the results, we found that the fluorescence intensities of cyclin D1, CD34, vimentin, and cytokeratin in the normal, sPL, and UCMSCs groups were significantly higher than in the model group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Western blot results showed that sPL and UCMSCs increased CK18 and stromal cell marker vimentin expression levels in a thin-endometrium rat model. Cyclin protein changes are a sign of cell proliferation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn comparison with the model group, cyclin D1 and CD34 expression were higher in the treatment group, but no significant differences were found between sPL and UCMSC treatments (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A higher level of cytokeratin and vimentin expression was observed in the sPL group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared to the blank normal group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). There were no statistically significant differences between the normal and sPL groups. A significant difference was observed in the expression of cytokeratin and vimentin between the subcutaneous and model groups (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Based on Western blot analysis, the experimental group showed significantly higher cytokeratin and vimentin expression than the normal group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, no significant difference was observed between the normal and experimental groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 sPL and UCMSCs promote the expression levels of PDGF-BB, E2, TGF-β1 and FSH\u003c/h2\u003e \u003cp\u003eCompared with the normal group, serum PDGF-BB and TGF-β1 levels in the model groups were increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), E2 and FSH levels were decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). After 21 days or 42 days of treatment, compared with the model group, the levels of the PDGF-BB, TGF-β1, E2 and FSH in the sPL group and UCMSCs group were decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Based on (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), there was no statistical difference between the two groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eIt has been estimated that the endometrium undergoes more than 400 cycles of development, growth, and shed during a woman's reproductive years. Endometrial thinness occurs as a result of inflammation, endometrial dysfunction, and uterine surgery. To gain a better understanding of the mechanisms underlying endometrial injury and repair, this study investigated the effects of sPL and UCMSCs on thin endometrium. Using histomorphology, Western blotting, and immunocytochemistry to diagnose and repair the damaged microenvironment. It is well-established that absolute ethyl alcohol perfusion in rats induces endometrial injury, Offering advantages such as easy modeling and high success rates. Several in vivo experiments demonstrated that the morphology and thickness of the endometrium were both significantly improved by sPL and UCMSCs. In previous studies, UCMSCs have been shown to thicken the endometrium, increase glands, and increase sub-endometrial angiogenesis, all of which improve pregnancy rates [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The therapeutic effectiveness of sPL significantly exceeded that of UCMSC transplantation in rats with thin endometrium by enhancing endometrial thickness, morphology, function, and repair capacity. Our study suggests that sPL combined with UCMSC therapy may be a promising approach for treating thin endometrium.\u003c/p\u003e \u003cp\u003eThis study suggests that intrauterine administration of autologous PRP has proliferative and anti-fibrotic effects on damaged endometrium. PRP contains growth factors and cytokines contributing to rapid healing and tissue regeneration by accelerating cell proliferation, angiogenesis, and migration [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, PRP has not been studied specifically on the endometrium. However, its major growth factor, PDGF, has been demonstrated to play several pivotal roles in endometrial cell proliferation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Diverse growth factors such as EGF, FGF, TGF-β, insulin-like growth factors, and colony-stimulating factors, along with their corresponding receptors, have been recognized for their participation in autocrine and paracrine functions, influencing endometrial cell growth, differentiation, and implantation [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMoreover, to the extent of our knowledge, this study is the first to reveal that autologous sPL can enhance endometrial proliferation, decrease fibrosis, and mitigate intrauterine adhesions by using a thin endometrial model. In a recent study, SPL has been demonstrated to have an anti-fibrotic effect on skeletal muscle healing after injury [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, the result of this study showed that sPL inhibited excessive collagen deposition, which may decrease fibrosis progression in thin endometriums. Compared with the model group, the thickness of the endometrium and the number of glands in sPL or UCMSC treatment groups were significantly higher, and the interstitial blood vessels were denser.\u003c/p\u003e \u003cp\u003eA significant increase in endometrial thickness and gland number was observed in the sPL-treated or UCMSC-treated groups, and interstitial blood vessels were decreased significantly. Vimentin and cytokeratin are often considered specific epithelial cell markers [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In endometrial cells, keratin and vimentin are important components of the intermediate filament protein cytoskeleton [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Their role is crucial in promoting cell proliferation and differentiation, and they are often considered dynamic structures [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In rat endometrial epithelial cells and luminal epithelial cells, cytokeratin 18 is predominantly found in the cytoplasm [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Within the mesenchyme cytoplasm of rats, vimentin is predominantly expressed in stromal cells [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Rat endometrium was found to have high levels of CK18 and vimentin after intrauterine perfusion with sPL and UCMSC. Microscopic examination reveals an intact and tight arrangement of the glandular epithelium and stroma of the endometrium due to changes in endometrial thickness and cyclin D1, CD34, CK18, and vimentin expression levels. Our research showed that sPL and UCMSC perfusion could restore endometrial thickness after intrauterine perfusion and facilitate the rejuvenation of glandular epithelial cells and stromal cells in a thin endometrial structure.\u003c/p\u003e \u003cp\u003eAccording to previous studies, estrogen and progesterone regulate VEGF expression in the endometrium, transforming it from thin and dense to thick and transparent [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. VEGF could influence the endometrium's proliferative, secretory, and shedding in combination with female hormones [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Low levels of VEGF in the menstrual phase are associated with endometrial shedding [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In contrast, high levels of VEGF in the secretory phase promote neovascularization and vascular permeability, both of which are beneficial to endometrium repair and embryo implantation during pregnancy [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In our study, VEGF was expressed throughout the implantation window in rat endometrium, suggesting that it could contribute to endometrial receptivity. Furthermore, SPL has also been applied to medical and surgical fields, including skin rejuvenation and hair regrowth, lung dysfunction, neural repair, and orthopedics due to its no-side effects properties.\u003c/p\u003e \u003cp\u003eIn conclusion, sPL and MSCs can significantly promote endometrium growth, offers significant benefits for thin endometrium treatments, providing a therapeutic option for thin endometrium. Despite ongoing challenges and difficulties, sPL shows significant promise for future clinical trials and could provide an alternative to PRP-based treatments. Furthermore, these promising results may contribute to the development of innovative therapeutic strategies for conditions associated with endometrial dysfunction. The combination of sPL and UCMSCs emerges as a potential candidate for clinical interventions aimed at enhancing endometrial regeneration and improving reproductive outcomes. sPL and MSCs hold great translational potential for regenerative medicine. We believe that further studies are necessary to determine the composite sPL\u0026thinsp;+\u0026thinsp;MSCs was generally superior to its corresponding monotherapy.\u003c/p\u003e"},{"header":"4. Materials and Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Animal Study\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e4.1.1 Animals\u0026rsquo; experiments\u003c/h2\u003e \u003cp\u003eSpecific pathogen-free Sprague-Dawley females\u0026rsquo; rats (aged 8\u0026ndash;10 weeks, weighing 220\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g) were purchased from Heilongjiang University of Chinese Medicine. All the Rats were housed in a specific virus-free laboratory at an ambient temperature of 23\u0026deg;C, and a 12-hour light/dark cycle and ad libitum access to food and water were provided after one week of acclimatization. The experiment was conducted on rats with complete sexual cycles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e4.1.2 sPL preparations\u003c/h2\u003e \u003cp\u003eAn average of 8 mL of blood was extracted from 20 anaesthetized rats by cardiac puncture and collected in 15 mL centrifuge tube containing 20 U sodium heparin. The red blood cells were removed, and the platelet was collected by centrifuging fresh blood at 1,500 g for 15 min. Platelet activation was achieved by incubating PRP with 20 mM CaCl\u003csub\u003e2\u003c/sub\u003e and 2 U/mL sodium heparin for 2 h at 37\u0026deg;C. Then, the mixture was frozen at -80\u0026deg;C for 2 h, followed by thawing at 37\u0026deg;C and cooling at 4\u0026deg;C to allow fibrin coalescence. sPL is collected from the platelet through ultra-low temperature freeze-thawing. After centrifugation at 3,000 g for 8 min, the supernatant was filtered through a 0.22 \u0026micro;m filter (Millipore, Darmstadt, Germany) to obtain the sPL. A total of 16 mL sPL was collected from 160 mL blood, the prepared and actived sPL was frozen and stored at -20\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Culture and amplification of UCMSCs\u003c/h2\u003e \u003cp\u003eThe UCMSCs were from the mothers who just gave birth, and informed consent was obtained from hospitalized mothers at The First Affiliated Hospital of Harbin Medical University, and rapidly processed. The Ethics Committee of Heilongjiang University of Chinese Medicine approved this study. All experiments were performed in accordance with relevant guidelines and regulations. The cells were harvested and cultured by centrifugation at 1,200 g for 10 min. The cells were resuspended and cultured in DMEM/F12 media with 10% fetal calf serum and 1% P/S at 37\u0026deg;C in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. Three times a week, the culture medium was changed. 0.05% trypsin-EDTA was used to harvest adherent UCMSCs at 90% confluence. After obtaining the third passage of culture, the UCMSCs were used for transplantation. Trypsin-EDTA was used to harvest the cells, followed by two washes with DMEM/F12 before resuspending them at 62,500 cells/\u0026micro;L in DMEM/F12.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Preparation of injection mixtures\u003c/h2\u003e \u003cp\u003eTo obtain a proper mixture, an efficient dispersion of the composition of the mixture must be provided to avoid precipitation. The chitosan powders were dissolved in 0.01 mol/L of acetic acid solution. The mixture was heated to 60\u0026deg;C under continuous magnetic stirring (200 rpm) for 180 min for better dissolution and cooling to 4\u0026deg;C. This can be accomplished through a three-way stopcock device, connecting a syringe with 160 \u0026micro;L sPL or UCMSCs on one side and another syringe with 40\u0026micro;L 5% chitosan (Sigma-Aldrich, St. Louis, USA) or extracellular matrix (ECM, 354263, BD Biosciences, Bedford MA, USA) on the opposite side. The device ensures a homogeneous mixture, consolidating components within a single syringe for injection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Model preparation and treatment\u003c/h2\u003e \u003cp\u003eThe estrus status of rats was monitored daily through vaginal smears. Nembutal sodium was used for general anesthesia during the operation on rats in stable estrus. Rats were placed in the supine position on the operating table, and the lower abdomen was scraped and disinfected with ethanol. The abdomen skin and muscles were deeply opened, starting with a longitudinal incision of 1.5 to 2 cm, followed by layer-by-layer openings. Near the vagina and ovary, the uterus was ligated. Then, 200\u0026ndash;300 \u0026micro;L PBS was kept in the uterus for 5 min in the normal groups and 200\u0026ndash;300 \u0026micro;L 95% ethanol was injected in the model and treatment groups for 5 min, respectively. In addition to extracting the remaining ethanol, the uterine cavities were washed twice with physiological saline. Three rounds of iodophor were applied to the incision site and sutured to return the uterus to its normal anatomical position after the incision was made.\u003c/p\u003e \u003cp\u003eThe normal group was maintained normally without intervention. Model rats were divided into five groups: Model group (40 \u0026micro;L 5% chitosan, 40 \u0026micro;L ECM and 120 \u0026micro;L PBS / rat, n\u0026thinsp;=\u0026thinsp;20), ECM\u0026thinsp;+\u0026thinsp;sPL group (40 \u0026micro;L ECM and 160 \u0026micro;L sPL / rat, n\u0026thinsp;=\u0026thinsp;20), ECM\u0026thinsp;+\u0026thinsp;cell group (40 \u0026micro;L ECM and 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e UCMSC/160 \u0026micro;L / rat, n\u0026thinsp;=\u0026thinsp;20), Gel\u0026thinsp;+\u0026thinsp;sPL group (40 \u0026micro;L 5% chitosan and 160 \u0026micro;L sPL/ rat, n\u0026thinsp;=\u0026thinsp;20), and Gel\u0026thinsp;+\u0026thinsp;cell group (40 \u0026micro;L 5% chitosan and 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e UCMSC/160 \u0026micro;L / rat, n\u0026thinsp;=\u0026thinsp;20). After modeling for 6 to 8 h, we perfused the therapeutic mixture into the uterus cavity with a medical syringe. To evaluate the endometrium status, rats were anesthetized for sampling at 21 days and 42 days after treatment. To further investigate, the uterus of rats was sectioned and preserved at -80\u0026deg;C or 4% paraformaldehyde.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Histopathological examinations\u003c/h2\u003e \u003cp\u003eThe rat uterus was removed in each group and placed in paraffin. The uterus was sliced at a thickness of 5 \u0026micro;m and dried at 45\u0026deg;C. The sections were covered in xylene and ethanol, varying from high to low concentrations. We stained the sections with HE stain kit (G1120, Solarbio), and used CaseViewer image processing software (3DHISTECH, Hungary) to analyze the vertical distance between the uterine cavity and the myometrium. A comparison of group differences in endometrial morphology and thickness was made by obtaining data from five random views of the endometrium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.6 Immunohistochemistry\u003c/h2\u003e \u003cp\u003eThe uterine was stored in 4% paraformaldehyde and placed in wax blocks. To block endogenous peroxidase activity and non-specific antigens, the sections were deparaffinized in xylene, rehydrated with ethanol, then incubated with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 15 min and 10% goat serum for 20 min. The sections were incubated with the primary antibodies CD34 (1: 2,500, ab81289, Abcam), cyclin D1 (1: 100, ab134175, Abcam), cytokeratin 18 (1: 500, ab133263, Abcam) and vimentin (1: 200, sc-6260, Santa Cruz) at a dilution of 1: 100 overnight at 4\u0026deg;C. Slides were incubated with 50 \u0026micro;L secondary antibodies and followed by 3,3\u0026prime;-diaminobenzidine tetrahydrochloride (DAB) solution for 1 h. A hematoxylin solution was then applied to the slides for 3 min, and the slides were analyzed with CaseViewer image processing software (3DHISTECH, Hungary).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.7 Western Blot analysis\u003c/h2\u003e \u003cp\u003eUterine tissues were lysed by RIPA lysis buffer to extract proteins. After lysing, the Uterine tissues in 400 \u0026micro;L lysis buffer (P0013C, Beyotime) and protein (30 \u0026micro;g / 20 \u0026micro;L) were stained with loading dye, separated with 8% \u0026minus;\u0026thinsp;12% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes and incubated with 5% skim milk. The membranes were exposed to cyclin D1 antibody (1: 2,000, ab134175, Abcam), CD34 antibody (1: 5,000, ab81289, Abcam), Pan-keratin antibody (1: 1,000, #4545, cell signaling technology), cytokeratin 18 antibody (1: 1,000, ab133263, Abcam) and vimentin antibody (1: 200, sc-6260, Santa Cruz) overnight. The membranes were washed with TBST and incubated with secondary anti-rabbit (1: 5,000, ab6721, Abcam) or anti-mouse IgG (1: 5,000, ab6789, Abcam) for 1 h at room temperature. The chemiluminescent signal was developed using the ECL Kit (Invitrogen, Waltham, MA, USA) and the protein contents was detected using ImageJ 1.52a (National Institutes of Health, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.8 Biochemical and Histopathological Analysis\u003c/h2\u003e \u003cp\u003eAll blood samples collected by abdominal aortic blood were stood at room temperature for 1 h, then the samples were centrifuged at 6,000 g for 15 min to collect the serums. The serums extracted from the blood samples of the experimental rats were separated and stored at -20\u0026deg;C. The concentrations of PDGF-BB, E2, TGF-β1 and FSH were performed using ELISA kits (R\u0026amp;D Systems, Inc., Minneapolis, MN, USA) according to the instructions provided by the manufacturer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.9 Ethics statement\u003c/h2\u003e \u003cp\u003e The animal study was reviewed and approved by the Ethical Committee for Animal Care and Use of the Heilongjiang University of Chinese Medicine (Ethics approval number: 2024011615). All authors confirm that all animal procedures and care were performed in accordance with relevant guidelines and regulations. All animal experiments were performed in accordance with the ARRIVE guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.10 Statistical analysis\u003c/h2\u003e \u003cp\u003eAnalysis was performed using the statistical software SPSS 22.0. The data in our study are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Students' T-tests, or a one-way analysis of variance (ANOVA), were used for comparisons between the two groups. Statistical significance was indicated by \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank to Ethical Committee for Animal Care and Use of the Heilongjiang University of Chinese Medicine for animal experiment support of this article (Ethics approval number: 2024011615).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.Q. M.\u0026dagger;: Conceptualization, Methodology, Software, Validation, Investigation, Data Curation, Writing \u0026ndash; Original Draft, Visualization; Y. Z.\u0026dagger;: Conceptualization, Methodology, Writing \u0026ndash; Original Draft; C.X. L.: Methodology, Validation, Data Curation; I.U.: Writing \u0026ndash; Review \u0026amp; Editing; T.Q. Z.: Data Curation; Y. Z.*: Conceptualization, Validation, Investigation, Supervision, writing \u0026ndash; review and editing. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData underlying the results presented in this paper are not publicly available at this time but may be obtained from the corresponding authors (Ling-Qi Meng: [email protected] and Yi Zhang: [email protected]) upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was received for conducting this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were performed in accordance with the ARRIVE guidelines. Approval was granted by the Ethics Committee of University of Chinese Medicine (Ethics approval number: 2024011615).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors gave consent for publication. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eORCID ID:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLing-Qi Meng\u0026dagger;: 0009-0007-8288-5111.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYi Zhang*: 0000-0002-4012-8258.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAAyhan, A. et al. Oncologic and obstetric outcomes of early-stage epithelial ovarian cancer patients who underwent fertility-sparing surgery: A retrospective study. \u003cem\u003eInt. 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Biology and therapeutic targeting of vascular endothelial growth factor A. \u003cem\u003eNat. Rev. Mol. Cell Biol.\u003c/em\u003e \u003cb\u003e24\u003c/b\u003e, 816\u0026ndash;834. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41580-023-00631-w\u003c/span\u003e\u003cspan address=\"10.1038/s41580-023-00631-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Thin endometrium, Super activated platelet lysate (sPL), Umbilical cord mesenchymal stem cells (UCMSCs), Endometrium regeneration","lastPublishedDoi":"10.21203/rs.3.rs-5870571/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5870571/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThis study aimed to establish a rat model of thin endometrium and investigate the effects of super-activated platelet lysate (sPL) and umbilical cord mesenchymal stem cells (UCMSCs) on the thin endometrium in rats.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThin endometrium models were induced by infusing absolute ethyl alcohol into the uteri of female Sprague-Dawley (SD) rats. Rats were randomly assigned to several groups (Normal, Model, Extracellular matrix (ECM)\u0026thinsp;+\u0026thinsp;sPL, ECM\u0026thinsp;+\u0026thinsp;cell, Gel\u0026thinsp;+\u0026thinsp;sPL, Gel\u0026thinsp;+\u0026thinsp;cell) and treated for 21 or 42 days. Histopathological structures and endometrial thickness were observed using hematoxylin-eosin (HE) staining. ELISA was used to detect PDGF-BB, TGF-β1, E2 and FSH expression levels in serum. Furthermore, Western blot and immunohistochemical staining were used to assess the expression levels of cyclin D1, CD34, pan-keratin, cytokeratin 18, and vimentin in uterine tissue.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHE staining revealed improvements in endometrial thickness, gland number, and blood vessels following treatment with sPL and UCMSCs in the thin endometrium rat model. Compared to the model group, ELISA results demonstrated that the PDGF-BB, E2, TGF-β1 and FSH serum in treatment groups returned to normal levels. Immunohistochemical staining and Western blot results indicated decreased keratin, cytokeratin, and vimentin expression levels in the model group, which were significantly increased by sPL perfusion or UCMSCs transplantation.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIntrauterine perfusion of sPL improves endometrium thickness, morphology, function, and repair capacity in rats with thin endometrium. The therapeutic efficacy of uterine infusion of sPL surpasses that of UCMSCs transplantation.\u003c/p\u003e","manuscriptTitle":"Therapeutic Potential of Super Activated Platelet Lysate (sPL) and Umbilical Cord Mesenchymal Stem Cells (UCMSCs) in Enhancing Endometrial Regeneration in Rats with Thin Endometrium","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-04 05:40:52","doi":"10.21203/rs.3.rs-5870571/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-21T05:03:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-15T23:53:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-07T07:17:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"221800155805541171445425606512031115441","date":"2025-04-24T19:58:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"158908670313782344575026009797401859388","date":"2025-04-23T16:51:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-23T02:51:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"301753819994129143329768019760855578328","date":"2025-04-23T01:22:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-18T12:09:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"282566859055416259354972108978667015446","date":"2025-04-06T06:07:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-03T08:36:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-31T00:37:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-31T00:36:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f9d3c9bf-ec97-43c6-aaf0-b99adc278d7d","owner":[],"postedDate":"April 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46622387,"name":"Biological sciences/Stem cells/Mesenchymal stem cells"},{"id":46622388,"name":"Health sciences/Diseases/Reproductive disorders/Infertility"}],"tags":[],"updatedAt":"2025-07-14T16:07:28+00:00","versionOfRecord":{"articleIdentity":"rs-5870571","link":"https://doi.org/10.1038/s41598-025-10587-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-09 15:58:00","publishedOnDateReadable":"July 9th, 2025"},"versionCreatedAt":"2025-04-04 05:40:52","video":"","vorDoi":"10.1038/s41598-025-10587-w","vorDoiUrl":"https://doi.org/10.1038/s41598-025-10587-w","workflowStages":[]},"version":"v1","identity":"rs-5870571","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5870571","identity":"rs-5870571","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}