Abstract: BACKGROUND: Research has found that human umbilical cord blood platelet rich plasma and human umbilical cord blood derived mesenchymal stem cells have certain therapeutic effects on thin endometrium. However, there are currently no reports on the study of human umbilical cord blood mononuclear cells on thin endometrium, and there is a lack of relevant research comparing the three.
OBJECTIVE: To explore the effects and mechanisms of human umbilical cord blood platelet rich plasma, monocytes, and mesenchymal stem cells in repairing thin endometrium in rats.
METHODS: Sixty female SPF grade SD rats were randomly divided into sham operation group, model group, human umbilical cord blood platelet rich plasma group, human umbilical cord blood mononuclear cell group, and human umbilical cord blood derived mesenchymal stem cell group, with 12 rats in each group. The sham operation group received 0.5 mL physiological saline injection into the uterine horn, followed by 0.5 mL of PBS infusion after 5 minutes; The model group, human umbilical cord blood platelet rich plasma group, human umbilical cord blood mononuclear cell group, and human umbilical cord blood derived mesenchymal stem cell group were injected with 0.5 mL of 95% ethanol by volume. After 5 minutes, the remaining ethanol was aspirated and washed twice with physiological saline. Then, 0.5 mL of PBS, human umbilical cord blood platelet rich plasma, human umbilical cord blood mononuclear cells (1×107 cells/mL), and human umbilical cord blood derived mesenchymal stem cells (1×107 cells/mL) were perfused separately. During the third normal estrus cycle after reperfusion, organs, tissues and serum were collected for testing of relevant indicators.
RESULTS AND CONCLUSION: (1) The macroscopic view of uterine tissue, hematoxylin eosin staining and Masson staining results: the sham operation group had intact structure, moderate endometrial thickness, and clear vascular structure. Compared with the sham operation group, the model group showed uterine atrophy, incomplete structure, significantly reduced endometrial thickness and glandular quantity, disordered vascular structure, and increased fibrosis. Compared with the model group, after treatment with human umbilical cord blood derivatives, the size, structure, and endometrial thickness of the uterus were restored (all P < 0.01), and fibrosis was reduced, with the most significant recovery observed in the human umbilical cord blood mononuclear cell group. The increase in glandular quantity was most significant in the human umbilical cord blood platelet rich plasma group (P < 0.000 1). (2) The immunohistochemistry and immunofluorescence results of uterine tissue showed that compared with the sham operation group, the expression levels of cell proliferation related indicators such as keratin 9 and vimentin, endometrial receptivity related indicators such as leukemia inhibitory factor and integrin αγβ3, platelet endothelial cell adhesion molecule, basic fibroblast growth factor, and vascular endothelial growth factor were all reduced in the model group (all P < 0.05). Compared with the model group, the above indicators were significantly increased after treatment with human umbilical cord blood derivatives. Comparison of human umbilical cord blood derivatives among groups showed that keratin 9 and vascular endothelial growth factor protein: human umbilical cord blood mononuclear cell group > human umbilical cord blood derived mesenchymal stem cell group > human umbilical cord blood platelet rich plasma group; Wave shaped protein and leukemia inhibitory factor protein: human umbilical cord blood derived mesenchymal stem cell group > human umbilical cord blood mononuclear cell group > human umbilical cord blood platelet rich plasma group; Integrin αγβ3 protein: human umbilical cord blood platelet rich plasma group > human umbilical cord blood derived mesenchymal stem cell group > human umbilical cord blood mononuclear cell group; Platelet endothelial cell adhesion molecule protein: human umbilical cord blood platelet rich plasma group > human umbilical cord blood mononuclear cell group > human umbilical cord blood derived mesenchymal stem cell group; Basic fibroblast growth factor protein: human umbilical cord blood mononuclear cell group > human umbilical cord blood platelet rich plasma group > human umbilical cord blood derived mesenchymal stem cell group. (3) Western blot analysis showed that compared with the sham operation group, the protein levels of interleukin-6, interleukin-1β, and tumor necrosis factor alpha in the model group were significantly increased (all P < 0.001), and their expression levels decreased after treatment (all P < 0.05). (4) ELISA assay showed that compared with the sham operation group, the model group had lower levels of anti Mullerian hormone, estradiol, and progesterone, and increased levels of follicle stimulating hormone and luteinizing hormone (except for luteinizing hormone, all P < 0.05). After treatment, there was a certain degree of recovery in the levels of sex hormones and anti Mullerian hormones. (5) Fertility experiments showed that compared with the sham operation group, the model group had an increase in conception time and a significant decrease in litter size (all P < 0.05). After treatment with human umbilical cord blood derivatives, the litter size of all three groups increased (P < 0.05), and no significant differences were found between the groups. This study preliminarily reveals that human umbilical cord blood mononuclear cells have a certain therapeutic effect on thin endometrium, and human umbilical cord blood platelet rich plasma, human umbilical cord blood mononuclear cells, and human umbilical cord blood derived mesenchymal stem cells have different advantages and differences in improving endometrial regeneration function, endometrial receptivity, angiogenesis, inflammation regulation, and improving pregnancy outcomes in thin endometrium.

Key words: human umbilical cord blood platelet-rich plasma, human umbilical cord blood mononuclear cell, human umbilical cord blood derived mesenchymal stem cell, thin endometrium, SD rat