8-week-old sexually mature and unmated C57BL/6 female mice (SPF grade, Chengdu Lilai Biotechnology Co., Ltd, China) were used in the present study. They were housed three to four rats per cage at room temperature and given ad libitum access to a standardized diet and tap water. The content of this experiment involving animal experiments has been approved by the experimental Animal Management and Ethics Committee of West China Second Hospital of Sichuan University.

The mice were divided into 3 groups after being adaptively fed for 7 days: the control group (n = 7), open the abdominal cavity and pull out the uterus without other surgical treatment; IUA model group (n = 10), mechanical injure the uterine horns and inject 30 µl PBS via vagina at 7th, 11th, and 15th day after the operation and the process of IUA modeling was shown in Fig. 1; MenSCs treated group (n = 10), mechanical injure the uterine horns and inject 30 µl MenSCs suspension (1 × 107/ml) via vagina at 7th, 11th, and 15th day after the operation. Four days after treatment, harvest the uterine tissues for morphological comparison and further examination. The specific grouping arrangements are shown in Table 1, and the transplantation treatment process is shown in Fig. 2. The mice were anesthetized by intraperitoneal injection of 30 mg/kg sodium pentobarbital before the experimental invasive procedure. After the end of the experiment, all the mice were anesthetized, and then the cervical vertebrae were dislocated and died.

The schematic diagram for IUA modeling process. (A): A 1.5 cm longitudinal incision was made in the lower abdomen to expose the uterus; (B): Observe the morphology of the uterus and exclude mice with uterine abnormalities; (C): Scrape the uterine cavity in a clockwise direction with a uterine scraper; (D): Suture the abdominal skin and subcutaneous tissue

Transplantation treatment process. (A): intravaginal injection of MenSCs suspension; (B): post-administration treatment to prevent leakage

Menstrual blood in the first four days was collected with a sterile menstrual cup and stored in a sterile tube with a preservation solution at 4℃. Inclusion criteria for menstrual blood are healthy females without vaginal discharge or infection, negative for HBV and HCV infection, and age ranges between 20 and 35 years old. Informed consent was obtained for every participant. 5 ml menstrual blood was decanted into an isolation buffer containing 2.5 µg/ml fungizone, 1% Pen/Strep, and 0.5 mM EDTA in 20 ml PBS. Then slowly added the blood sample into Ficoll-paque (No 17544202; Cytiva) of the same volume and centrifuged at 400 g for 20 min. The turbid interface phase of Ficoll and cell culture media was added in 3 ml PBS and centrifuged at 100 g for 10 min. The cell pellet was resuspended in DMEM-F12 (No. 10565018; Gibco), 10% FBS (No. 10099141c; Gibco), 1% Pene/Strep (No. 15140122; Gibco), 1% glutamine (No. 35050061; Gibco) at 37 ℃ in a 5% CO2 humidified incubator. The medium was refreshed every 2 days. Passaging was performed with 0.5% trypsin every 2 days when cell density reached 90%. Take the third-generation cells to record growth morphology and characterization.

To confirm MenSCs identity, collect single-cell suspension using EDTA and resuspend them at a concentration of 1 × 107 cells/ml in BD Pharmingen™ Stain Buffer (No. 554656; BD Bioscience). Cells were stained with a Human MSC Analysis Kit (No 562245. BD; USA) according to the manufacturer’s instructions and CD surface antigens were analyzed using a CytoFLEX LX (Beckman Coulter).

For MenSCs characterization, cells were plated on glass bottom culture dishes. When the cell density reached approximately 90%, MenSCs were fixed in 4% paraformaldehyde at 2–8℃ for 15 min and blocked with 10% goat serum for 30 min. Then, the cells were incubated with primary antibodies at 4℃ and in secondary antibodies for 1 h at room temperature. The antibodies used are as follows: Integrin beta 1 (ITGB1, No. ab179471; Abcam), CD105 (No. ab221675; Abcam), CD44 (No. ab243894; Abcam), CD90 (No. ab307736; Abcam), and Goat anti-Rabbit Alexa Fluor™ 555 (No. A32732; Invitrogen). After washing with PBS, incubate the cells with DAPI and cover them with an antifade mounting medium (No. H-1000; VECTASHIELD). For endometrial markers detection, each group of uterine tissue, after fixation and dehydration, was embedded in OCT and cut into sections of 10 μm in thickness. After washing with PBS, the slices were blocked with 10% goat serum and 0.1% Triton X-100 in PBS at room temperature for 1 h. After block and permeabilization, the sections were immersed in anti-Cxcl13 (No. 15782; ABclonal) and anti-Cxcr5 (No. A8950; ABclonal) at 2–8℃ overnight. Goat anti-Rabbit Alexa Fluor™ 555 (No. A32732; Invitrogen) was incubated with cells at room temperature for 1 h. A confocal microscope (Leica SP8) was used to collect the images. The fluorescence intensity was analyzed by ImageJ software.

A segment of the uterus in the same position was taken and fixed in 4% paraformaldehyde for more than 24 h, embedded in paraffin, and sliced. Xylene dewaxed for 5 min, dehydrated with different concentrations of ethanol, stained with hematoxylin staining solution for 5 min, differentiated for 30 s, soaked in 1 × PBS for 15 min, soaked in eosin for 1 min, dehydrated with different concentrations of ethanol, treated with xylene transparent and fixed with paraffin. The morphology of the endometrium was observed under a microscope, and the thickness of the endometrium, the number of glands and endometrial blood vessels were calculated with Image pro plus 6.0.

The sections were conventional dewaxed and stained with Weigert ferritin dye, ponceau acid fuchsin staining solution, and Aniline blue staining solution, respectively. Different concentrations of ethanol were used for dehydration and xylene transparent treatment. After fixation, the blue staining area ratio of endometrial fibrosis was calculated by Image Pro plus 6.0 under a microscope.

MiRNeasy Mini Kit (No. 160040742; QIAGEN) was used to extract total RNA from the uterus. RNA concentration and purity were measured by NanoDrop 2000 C spectrophotometer. The total RNA samples were reversely transcribed using a cDNA Synthesis Kit (No. R211-01; Vazyme). The amplification system was prepared according to the specification step by Taq Pro Universal SYBR qPCR Master Mix (No. Q712-02; Vazyme). The dissolution curve and CT value results were derived after amplification, and three experiments were carried out repeatedly. The relative gene expression levels were calculated using the 2−△△CT method. The primers used for the RT-qPCR analysis are presented in Table 2.

GraphPad Prism 8.0 was used for statistical processing. Data were expressed as mean ± SD. Statistical comparisons among groups were performed with one-way ANOVA. P < 0.05 were considered statistically significant. The work has been reported in line with the ARRIVE guidelines 2.0.