A Novel Method to Repair Thin Endometrium and Restore Fertility Based on Menstruation-Derived Stem Cell

Abstract

Thin endometrium (TE), which mainly occurs as a result of severe damage to the endometrial basalis, is one of the prominent etiologies of menstrual abnormalities, infertility, and recurrent miscarriage in women. Previous studies have demonstrated that mesenchymal stem cells (MSCs) are considered ideal cells with multipotency for regenerative medicine and exhibit therapeutic effects on TE through their cellular secretions. However, there is limited research on strategies to enhance MSC secretion to improve their therapeutic efficacy. Herein, we isolated menstrual blood-derived mesenchymal stem cells (MenSCs) from menstruation and transformed them into decidualized stromal cells (DSCs), which are specialized cells with enhanced secretory functions. To assess the therapeutic potential of DSCs compared to MenSCs, we conducted a series of experiments in cells and animals. The results demonstrated that DSCs exhibited changes in morphology compared to MenSCs, with a decrease in cell proliferation but a significant improvement in secretion function. Furthermore, DSCs facilitated the restoration of endometrial thickness and increased the number of glands and blood vessel formation. Most importantly, the pregnancy rates in rats were effectively restored, bringing them closer to normal levels. These findings greatly contribute to our understanding of stem cell therapy for TE and strongly suggest that DSCs could hold significant promise as a potential treatment option for TE.

Keywords: Animal model; Decidualization; Menstrual blood stem cells (MenSCs); Stem cell therapy; Thin endometrium.

Fig. 1

Isolation and identification of MenSCs. A The morphology of MenSCs in the bright-field. Scale bar: 210 μm. B Cell composition in primary cell (P0), passage 1 (P1), and passage 2 (P2). Green represents the stromal cell marker (VIM), red represents the epithelial cell marker (CK7), and blue represents the nuclear marker (DAPI). Scale bar: 170 μm. C Isolated MenSCs at passage 4 were used for flow cytometry analyses, and the values represent the percentage of positive cells among all cells. CD44 (99.54%), CD73 (98.30%), CD90 (91.88%), CD105 (92.88%), CD11b/CD34/CD79a (0.04%). (D) Immunofluorescence staining verified the multipotent differentiation ability of the MenSCs, representing the ability of adipogenesis (FABP-4), osteogenesis (osteocalcin), and chondrogenic (aggrecan) differentiation, negative control was undifferentiated MenSCs. Negative control scale bar: 50 μm. Multipotential differentiation scale bar: 170 μm. E OCT-4 gene expression in the MenSCs, the control is fibroblast cells. F OCT-4 protein in the fibroblast (negative control) and MenSCs. Scale bar: 50 μm

Fig. 2

MenSCs induced into decidual stromal cells. A Schematic overview of MenSCs/DSCs treatment for thin endometrium. B The morphology of MenSCs and decidual stromal cells (DSCs) in the bright-field: (i) Cell morphology on the 1st of the control group, (ii) cell morphology on the 14th of the control group, (iii) cell morphology on the 1st of the decidualization group, (iv) cell morphology on the 14th of the decidualization group, Scale bar: 210 μm. C Cell proliferation curve between MenSCs and DSCs. D Expression of decidual-related genes (PRL and IGFBP-1) and endometrial receptivity-related genes (LIF and HOXA-10) after MenSCs-induced decidualization. E The PRL concentration in the supernatant was measured in different groups, with the control group being the DMEM medium

Fig. 3

MenSCs and DSCs secret more VEGF-A in vitro. A The concentration of soluble VEGF-A in control (un-conditioned media), MenSCs (MenSCs supernatant), and DSCs (DSCs supernatant). B The impact of supernatants from different groups on the proliferation and migration of vascular endothelial cells was investigated. Scale bar: 530 μm. C Quantification of the wound healing assay results. Values are expressed as average ± SEM of three replicates

Fig. 4

The therapeutic effects of MenSCs and DSCs on TE rats. A Expression of genes after treatment in different groups. VEGF-A: angiogenesis-related gene. B Expression of uterine vascular endothelial (CD31) in rats among different groups. Control, without any treatment; TE, rat model of thin endometrium; TE+MenSCs, MenSCs treat TE; TE+DSCs, DSCs treat TE. Scale bar: 170 μm. C H&E staining of rat uterine after different treatments. Scale bar: 100 μm. D Statistical analysis of endometrial thickness after different treatments. Values are expressed as average ± SEM of three replicates. E Statistical analysis of glands of the endometrium after different treatments. Values are expressed as average ± SEM of three replicates. F Image of embryo numbers on each side of the uterus after treatment (control vs sham), and statistical analysis of the embryo numbers. G Image of embryo numbers on each side of the uterus after treatment (sham vs TE), and statistical analysis of the embryo numbers. H Image of embryo numbers on each side of the uterus after treatment (sham vs TE+MenSCs), and statistical analysis of the embryo numbers. I Image of embryo numbers on each side of the uterus after treatment (sham vs TE+DSCs), and statistical analysis of the embryo numbers. *p < 0.05, **p < 0.01, ***p < 0.001