Menstrual blood-derived mesenchymal stem cells combining with platelet-rich plasma infusion in endometrium repair

To determine the effect of MenSCs with or without PRP co‐cultivation in ameliorating endometrial injuries, we observed the histological changes and measured the endometrial thickness in mice challenged with ethanol. As shown in Figure  1a, b , we observed impaired endometrial structures in the model group compared to the control, including reduced uterine thickness, decreased gland numbers, and damaged endometrial cavity epithelial and glandular epithelial cells. When treated with MenSCs or PRP alone, although the pathological injuries of the uterus were significantly improved, the damages could not fully recover (Figure  1d, e ). Interestingly, in the MenSCs + PRP group, we observed a pronounced improvement in the endometrial structure and cellular injuries compared to the model group (Figure  1c ), which had the most significant effect among the MenSCs and PRP groups (Figure  1d, e ). MenSCs + PRP treatment significantly improved histological damages and thickness of mice endometrium challenged with ethanol. The morphology of endometrial tissue sections was observed after HE staining (red lines indicate the measurement standards for the endometrial thickness). The structure (b) and thickness (f) of endometrium in the model mice were markedly impaired comparing with control (a). The MenSCs (c), PRP (d), and MenSCs + PRP (e) all improved these damages and increased endometrial thickness (f), with MenSCs+ PRP showing the most significant efficacy ( n  = 3, # p  < 0.05, ## p  < 0.01, ### p  < 0.001, #### p  < 0.0001 vs. control; * p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001 vs. model; & p  < 0.05, && p  < 0.01, &&& p  < 0.001, &&&& p  < 0.0001 vs. MenSCs + PRP). Comparing with control, the endometrium thickness in the model mice significantly decreased (Figure  1f , p  < 0.0001). After treatments, the endometrium thickness was significantly increased in MenSCs, PRP, and MenSCs + PRP groups (Figure  1f , p  < 0.0001, p  < 0.0001, and p  < 0.0001 vs. model), with the MenSCs + PRP displaying the most pronounce efficacy compared to MenSCs or PRP alone ( p  = 0.0282 vs. MenSCs, p  < 0.001 vs. PRP). To further evaluate the potential effect of MenSCs + PRP, we determined the expression levels of CK18, vimentin, and ItgαVβ3 in response of endometrial growth and repair. As shown in Figure  2a–c , we observed that CK18 bound to the keratin in the cytoplasm of the endometrium epithelial cells, vimentin specifically expressed in the endometrial stroma cells, while ItgαVβ3 was both expressed in the lumen or glandular of endometrial epithelium cells and in the endometrial stroma. The expression of CK18, vimentin, and ItgαVβ3 were significantly decreased after ethanol perfusion compared to control (Figure  2d–f , all p  < 0.0001 vs. control). Comparing with model, the monotherapy of MenSCs and PRP increased CK18 (Figure  2d , both p  < 0.0001 vs. model), vimentin (Figure  2e , p  < 0.0001 and p  = 0.0009 vs. model), and ItgαVβ3 expression (Figure  2f , p  < 0.0001 and p  = 0.0117 vs. model), while the MenSCs + PRP therapy showed the most significant effect in upregulating the expressions of these proteins (Figure  2c, d , all p  < 0.0001 vs. model). Comparing with MenSCs therapy, the MenSCs + PRP combined treatment elevated CK18 expression more significantly (Figure  2d , p  = 0.0124 vs. MenSCs). Also, MenSCs + PRP therapy increased CK18, vimentin, and ItgαVβ3 expressions more pronouncedly comparing with PRP therapy alone (Figure  2d–f , p  < 0.0001, p  < 0.0001, and p  = 0.0024 vs. PRP). MenSCs + PRP significantly induced the protein expression of CK18, vimentin, and ItgαVβ3 in endometrium. The expressions of CK18 (a), vimentin (b), and ItgαVβ3 (c) were observed using IHC staining (red arrows indicate the positive areas in the immunostaining images), and the average optical density was measured using ImageJ software (d–f). The expressions of CK18, vimentin, and ItgαVβ3 were significantly decreased after ethanol perfusion. MenSCs, PRP, and MenSCs + PRP treatment enhanced CK18, vimentin, and ItgαVβ3 expressions, with MenSCs + PRP showing the most significant effects ( n  = 3, # p  < 0.05, ## p  < 0.01, ### p  < 0.001, #### p  < 0.0001 vs. control; * p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001 vs. model; & p  < 0.05, && p  < 0.01, &&& p  < 0.001, &&&& p  < 0.0001 vs. MenSCs + PRP). It is reported that the levels of CK18, vimentin, ItgαVβ3, and VEGF can be used to reflect the growth, repair status, and regenerative capacity of the endometrium. 17 , 18 , 19 , 20 We then determined the protein expressions of CK18, vimentin, and VEGF by WB. We found that vimentin (Figure  3a ), CK18 (Figure  3b ), and VEGF (Figure  3c ) expressions were all inhibited in the model group of mice (all p  < 0.0001 vs. control). After MenSCs, PRP, and MenSCs + PRP treatments, the protein expression of vimentin (Figure  3a , p  = 0.0046, p  = 0.0219, and p  = 0.003 vs. model), CK18 (Figure  3b , p  < 0.0001, p  < 0.0001, and p  = 0.0051 vs. model), and VEGF (Figure  3c , p  < 0.0001, p  < 0.0001, and p  = 0.0017 vs. model) were all upregulated. Surprisingly, comparing with MenSCs and PRP therapies, the MenSCs + PRP combined treatment showed more obvious effects in increasing CK18 (Figure  3b , p  = 0.0203 vs. MenSCs, p  = 0.0002 vs. PRP) and VEGF levels (Figure  3c , p  = 0.0037 vs. MenSCs, p  < 0.0001 vs. PRP). MenSCs + PRP significantly increased vimentin, CK18, and VEGF expressions in endometrium. The protein expressions in endometrium tissues were determined using WB assay. The vimentin (a), CK18 (b), and VEGF (c) levels were all markedly decreased after modeling, while were significantly increased by MenSCs + PRP, MenSCs, and PRP treatments, with MenSCs + PRP showing the most significant effects ( n  = 3, # p  < 0.05, ## p  < 0.01, ### p  < 0.001, #### p  < 0.0001 vs. control; * p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001 vs. model; & p  < 0.05, && p  < 0.01, &&& p  < 0.001, &&&& p  < 0.0001 vs. MenSCs + PRP).

Thin endometrial thickness, induced by various causes, remains a challenging problem in infertility management. Despite the application of current clinical medications, there is still a lack of promising and effective approaches. Therefore, finding new strategies to improve endometrial conditions is needed. The structure of the endometrium can be classified into two layers: the functional layer and the basal layer. The functional layer undergoes periodic shedding during the menstrual cycle and is expelled with menstrual blood, while the basal layer has robust proliferation and regenerative capabilities. This allows it to remain intact throughout the menstrual period and regenerate new functional layers. 21 MenSCs are endometrial stem/progenitor cells that originate from menstrual blood. They exhibit high proliferative activity and differentiation potential, similar to other types of stem cells. 22 The application of MenSCs is becoming a novel approach for treating endometrial diseases. Autologous MenSC transplantation has already been found to increase endometrial thickness in women with severe Asherman's syndrome. 9 In our previous study, we confirmed that MenSC transplantation was able to significantly improve endometrial thickness, with the best effect compared to bone marrow mesenchymal stem cells and umbilical cord‐derived mesenchymal stem cells. 23 Despite the significant effect of MenSCs in improving endometrial function, we observed that the level of restoration had not yet reached normal levels. Therefore, we aim to further identify novel methods that can enhance the efficacy of MenSCs. In recent years, a growing number of studies have indicated that intrauterine PRP infusion has the property to increase endometrial receptivity and thickness. This is beneficial for increasing pregnancy rates and is being explored as a new approach. 24 In an intrauterine mechanical injury model of rats, Zhang et al. reported that PRP improves the effect of MenSCs in treating intrauterine adhesions. 25 Additionally, recent studies have reported that MenSCs loaded with PRP are more effective in ameliorating chronic rotator cuff tears and differentiating into insulin‐producing cells. These findings indicate the potential ability of PRP to enhance the functions of MenSCs. 26 Thus, we wonder if MenSCs in combination of PRP infusion could improve the therapeutic effect in treating ethanol‐induced endometrial injuries furthermore. According to our previous reports, 15 s of 95% ethanol perfusion was sufficient to induce endometrial injury in mice, as ethanol treatment for less than 15 s did not result in significant damage, whereas treatment exceeding 15 s caused excessive damage to the uterus. In the present study, we investigated the effect of MenSCs + PRP in comparison with MenSCs or PRP alone in an ethanol‐induced endometrial injury model of mice. As we expected, the amelioration of endometrial damages and increase in thickness by MenSCs + PRP were the most significant among all groups, indicating a better therapeutic effect of MenSCs + PRP. The endometrium is composed of epithelial cells and stromal cells. The epithelial cells primarily include luminal epithelial cells, glandular epithelial cells, and vascular endothelial cells. 27 CK18, vimentin, ItgαVβ3, and VEGF are expressed in the epithelial cells, stromal cells, and vascular endothelial cells of the endometrium. The expression levels of these markers can be used to reflect the growth, repair status, and regenerative and reparative capacity of the endometrium. 17 , 18 , 19 , 20 In the following investigations, we measured the protein expressions of CK18, vimentin, ItgαVβ3, and VEGF using IHC staining and WB assays. The results suggested that MenSCs in combination with PRP are more effective in upregulating CK18, vimentin, ItgαVβ3, and VEGF levels, indicating their greater advantages in promoting endometrial tissue growth and repair. In conclusion, MenSCs treatment in combination with PRP infusion significantly ameliorated ethanol‐induced endometrial injuries by upregulating the expressions of CK18, vimentin, ItgαVβ3, and VEGF, which facilitated tissue growth and repair. The MenSCs + PRP combination showed the most significant efficacy compared to MenSCs or PRP alone. Therefore, combining MenSCs with PRP might be a feasible approach with a better therapeutic effect for treating thin endometrium. Future studies could potentially explore the optimal dosage and duration for MenSCs + PRP treatment, as well as investigate any potential unknown detrimental effects on embryo development, prior to conducting clinical trials.

The uterine cavity and endometrial conditions are critical factors influencing the conception rate and incidence of infertility. When the endometrial thickness is below 7 mm, referring to a thin endometrium, implantation of the embryo is severely affected. 1 It is reported that multiple intrauterine surgeries, long‐term use of contraceptive drugs, vaginal microbiota disturbance can alter endometrial receptivity. 2 Although the clinical application of medicines such as aspirin, estrogen, luteal estradiol, sildenafil citrate, and granulocyte colony‐stimulating factor is beneficial to thin endometrium, the efficacy of these drugs varies among different individuals and often requires long‐term medications. 3 Therefore, there is an urgent need to explore new strategies with promising effects in endometrial repair. As a highly regenerative tissue, the endometrium undergoes periods of growth and shedding during the menstrual cycle. An impaired capacity to rebuild normal uterine structures may contribute to the development of a thin endometrium. 4 The emergence of regenerative medicine has sparked growing interest among researchers in stem cell‐based therapies. Stem cells, as undifferentiated cells, possess a remarkable ability to repair tissue damage through the process of redifferentiation. 5 A growing number of studies have demonstrated the regenerative properties of bone marrow, endometrium, and menstrual blood‐derived stem cells in thin endometrial treatments. 6 , 7 Menstrual blood‐derived mesenchymal stem cells (MenSCs) can also be a source of stem cells. MenSCs have their own advantages owing to their non‐invasive nature, high proliferation rate, immune functions, and lack of ethical dilemmas. 8 Clinical study has proven that auto‐transplantation of MenSCs significantly increase endometrium thickness to 7 mm in female patients with Asherman's syndrome, which leads to successful conceiving and spontaneous pregnancy. 9 In vitro studies reveal that activating PI3K/Akt pathway 10 and Hippo/TAZ pathway, 11 increasing vimentin, vascular endothelial growth factor and keratin expression, 12 inducing epithelial differentiation, 13 regulating immune network 14 might be involved in the underlying mechanism of MenSCs. Interestingly, recent studies found that endometrial mesenchymal stem cells co‐culturing with platelet‐rich plasma (PRP) displayed significantly stronger growth, migration, and adhesion properties comparing with normal stem cells, suggesting that combination of PRP and stem cells may be a better approach to promote endometrial repair. 15 , 16 However, there are currently no studies reporting the effects of a combination treatment using PRP and stem cells, particularly PRP‐MenSCs, in the treatment of thin endometrium due to various causes. Therefore, we aimed to determine the effect of PRP‐MenSCs in ameliorating endometrial damage in the present study. In this study, we established an endometrial injury model in mice by infusing 95% ethanol into the uterine cavity, followed by in situ transplantation of MenSCs, PRP, or PRP‐MenSCs. On the third estrus cycle following the surgery, the endometrial tissues were collected for further investigation. We found that the endometrial thickness was significantly increased in the MenSCs, PRP, and PRP‐MenSCs groups. Our findings revealed significant improvements in the pathological injuries of endometrial tissues in these groups, with the PRP‐MenSCs group exhibiting the most remarkable effect. The protein expression of cytokeratin 18 (CK18), vimentin, ItgαVβ3, and VEGF was notably elevated after PRP‐MenSCs treatment. Therefore, PRP‐MenSCs may represent a novel approach for treating thin endometrium with promising efficacy and substantial potential for practical applications.

The authors report there are no competing interests to declare.

Chloral hydrate (C104202, Shanghai Aladdin Biochemical Technology Co., Ltd); 10% neutral formalin (G2161, Beijing Solaibao Technology Co., Ltd); DMEM/F12 (11320033, Gibco Life Technologies Co., Ltd); αMEM (41061029, Beijing Solarbio Technology Co., Ltd); 1% cyanine/streptomycin (15070063, Beijing Solarbio Technology Co., Ltd); 0.25% trypsin (25200072, Beijing Solarbio Technology Co., Ltd); CK18 primary antibody (66187‐1‐Ig, Proteintech Technology Co., Ltd); vimentin primary antibody (A19607, Wuhan ABclonal Co., Ltd); ItgαVβ3 primary antibody (abs136335, Shanghai absin Co., Ltd); VEGF primary antibody (abs131208, Shanghai absin Co., Ltd); Goat Anti‐Mouse IgG (A21010, Beijing Abbkine Co., Ltd); Goat Anti‐Rabbit IgG (A23240, Beijing Abbkine Co., Ltd), BSA kits (062122220830, Beyotime Biotechnology). After collecting menstrual blood from healthy women using sterile cups, it was diluted with PBS buffer containing antibiotics. The supernatant was then discarded by centrifugation, and the sediment at the bottom is collected. The sediment was mixed with complete culture medium (fetal bovine serum with 1% cyanine/streptomycin in DMEM/F12 solution) and transferred to a culture bottle for cultivation. During the cultivation process, non‐adherent cells were gradually discarded. The adherent cells were used as the MenSCs in the following investigations. The experiment protocol was approved by the ethics committee of the Xingtai People's Hospital (No. 2022[041]). To obtain PRP, whole blood samples from female was centrifuged at 2400 r/min for 10 mins. The supernatant and buffy coat were collected together with the uppermost 2 mm of the red blood cell layer. After a secondary centrifugation, the top three‐quarters of the suspension and the remaining red blood cell layer were removed. The collected material was then centrifuged at 3600 r/min for 15 mins. Three‐quarters of the supernatant were discarded, and the remaining liquid was gently mixed to resuspend the bottom layer, resulting in PRP. DMEM culture medium was added to the PRP at a 1:1 ratio and stored refrigerated. Prior to the experiment, a procoagulant (60 μL thrombin + 1 mL 10% calcium chloride) was added to the PRP at a ratio of PRP: procoagulant = 10:1 for subsequent administration. The MenSCs + PRP solution was prepared by co‐culturing 1 × 10 7 /mL MenSCs with PRP in 96‐well plates at a ratio of 2:1. The experiment protocol was approved by the ethics committee of the Xingtai People's Hospital (No. 2022[041]). Forty‐five healthy unmated Kunming SPF mice (30–35 g) were purchased from Hebei Experimental Animal Center (License: SCXK (Ji) 2018‐004). The animal experiment protocol was approved by the ethics committee of the Xingtai People's Hospital (No. 2022[041]). The animals were housed in 21 ± 2°C, 12 h light/12 h dark condition, with tap water and food ad libitum. After 7 days of adaptive feeding, the mice were randomly divided into control, model, MenSCs + PRP, MenSCs, and PRP groups. On the day of estrus in mice, surgical procedures were performed to establish an in vivo endometrial injury model. Mice in the experimental groups underwent laparotomy and intracavitary perfusion of 95% ethanol into the uterine. After anesthetizing and sterilizing, an incision was made approximately 1 cm above the urethral orifice to separate the uterus in mice. Hemostatic forceps were used to clamp the uterus near the vaginal end and near the ovarian end. The 95% ethanol was horizontally inserted from the vaginal end of the uterus. After infiltrating the liquid for 15 s in the uterus, saline solution was injected from the other end to irrigate the uterus. The same procedure was followed for the other side of the uterus. Subsequently, the MenSCs group of mice were transplanted with 0.1 mL MenSCs (2 × 10 6 –5 × 10 6 /mL) intracavitary, the PRP group received intracavitary administration of 0.1 mL PRP, and the MenSCs + PRP group was transplanted with 0.1 mL of MenSCs + PRP co‐culturing solution (2 × 10 6 –5 × 10 6 /mL). And the model group of mice were given with equal volume of PBS. The abdominal cavity was sutured to finish the modeling process. After the implantation, mice were continuously observed for three estrous cycles. Forty‐five mice were initially selected for the study. Following the assessment of their estrous cycles, 41 mice met the inclusion criteria and were included in the subsequent analysis. And during the estrus phase of the third cycle, the mice were euthanized, and uterine tissue was collected for subsequent investigations. The uterine tissue was fixed with 10% formalin, and a part of tissue was taken for gradient dehydration. It was then embedded in paraffin and sectioned, followed by staining with hematoxylin and eosin. The morphology of the endometrial cells was observed under microscope, and the thickness of the endometrium was measured using ImageJ software. The vertical distance from the junction of the endometrial muscle layer to the cavity of the uterus was measured at four random sites on each slide, and the average value was recorded as the endometrial thickness of the slide. The dewaxed tissue sections were subjected to antigen retrieval, followed by the addition of primary antibodies against CK18, vimentin, and ItgαVβ3. The sections were incubated overnight at 4°C. After washing off the primary antibodies, corresponding secondary antibodies and DAB were added for visualization. Subsequently, nuclear staining was performed using hematoxylin. The expression of CK18, vimentin, and ItgαVβ3 proteins in each section was observed under microscope. The average optical density values were quantified using ImageJ software. The endometrial tissue was subjected to cold lysis buffer for homogenization. The supernatant was collected, and the total protein concentration was determined using a BSA kit. The diluted protein samples were mixed with SDS‐PAGE loading buffer, followed by high‐temperature denaturation and separation on SDS‐PAGE gels of different concentrations. The proteins were then transferred from the gel to a PVDF membrane. The membrane was incubated with primary antibodies against CK18 (1:2000), vimentin (1:1000), VEGF (1:500), and β‐actin (1:2000) proteins overnight. The corresponding secondary antibodies (1:1000) were added to the membranes. And then, the membranes were incubated with high‐sensitivity enhanced chemiluminescence solution for protein grayscale analysis. In this study, data analysis was performed using SPSS 25.0 statistical software. Descriptive statistics for continuous variables are presented as mean ± standard deviation ( X −  ± SD). One‐way ANOVA was used for multiple group comparisons of continuous variables. Statistical analysis included a test for normality prior to using the mean and conducting one‐way ANOVA. The data was considered significant when a  = 0.05 and p  < 0.05.