CXCL12 Promotes Stem Cell Recruitment and Uterine Repair after Injury in Asherman's Syndrome

Abstract

Asherman's syndrome is an acquired condition of uterine fibrosis and adhesions in response to injury that adversely affects fertility and pregnancy. We have previously demonstrated that bone marrow-derived mesenchymal stem cells (BMDSCs) contribute to uterine repair after injury and that stem cells supplementation improves fertility. Here, we demonstrate that CXCL12 is the chemokine that mediates stem cell engraftment and functional improvement using a murine model of Asherman's syndrome. After uterine injury, we demonstrate that CXCL12 augmentation increased BMDSC engraftment and that the CXCL12 receptor (CXCR4) antagonist, ADM3100, blocked stem cell recruitment. CXCL12 reduced, whereas ADM3100 increased fibrosis. CXCL12 treatment led to improved fertility and litter size, whereas ADM3100 treatment reduced fertility and litter size. ADM3100 prevented optimal spontaneous uterine repair mediated by endogenous CXCL12 production, reducing pregnancies after injury in the absence of supplemental CXCL12 administration; however, ADM3100 treatment could be partially rescued by CXCL12 augmentation. CXCL12 or other CXCR4 receptor agonists may be useful in the treatment of infertility or adverse pregnancy outcomes in Asherman's syndrome and other related uterine disorders.

Keywords: AMD3100; Asherman’s syndrome; CXCL12; CXCR4; cell therapy; fertility; intrauterine adhesions; stem cells; uterus.

Figure 1

Immunofluorescence Staining of GFP, CD45, and Cytokeratin (A–C) Uterine tissue of GFP mice and spleen and skin tissue of wild-type mice were used as positive controls for GFP, CD45, and cytokeratin, respectively (A). Tissue sections from uteri of irradiated BM-transplanted mice are shown in (B) and non-irradiated, bone marrow-supplemented mice in (C). Tissues were stained with anti-GFP antibody (green) and co-stained with either anti-CD45 (pan-leukocyte marker) antibody (red) or cytokeratin (epithelial marker) antibody (yellow). Nuclei were stained by DAPI and are shown in blue. Fluorescence images were captured by confocal microscope. Here, we evaluate the recruitment of BM-derived stem cells after administration of GFP BM. Mice received either sham surgery (Sham) or induction of AS followed by administration of PBS control, CXCL12, AMD3100, or CXCL12 plus AMD3100. We evaluated the total number of BM stem cells (GFP⁺/CD45⁻) and the number of those cells that were contributing to the epithelial cell population (GFP⁺/CD45⁻/cytokeratin⁺) in the endometrium. The number of cells in each category is quantified in Figure 2.

Figure 2

Recruitment of Stem Cells into the Uterus of Asherman’s Syndrome (A) Irradiation for myeloablation and GFP⁺ BM transplant. (B) Intravenous GFP⁺ BM supplementation. The sham and AS groups were treated with PBS, CXCL12, AMD31000, or CXCL12 plus AMD3100. The presence of GFP⁺/CD45⁻ cells was used to determine the number of engrafted BM-derived stem cell. In addition, GFP⁺/CD45⁻/CK⁺ cells were indicative of BM-derived epithelial cells. Uterine sections were analyzed by immunofluorescence staining as demonstrated in Figure 1 and quantified here. As expected, the mice receiving full BM transplant had far more BM-derived cells engrafting the uterus compared to animals receiving only transient BM administration. In either model, uterine injury (AS) recruits far more BM-derived stem cells and BM-derived epithelial cells compared to uninjured controls. CXCL12 treatment results in greater numbers of BM-derived cells recruited. AMD3100 (a CXCR4 antagonist) blocks CXCL12-mediated stem cell recruitment. AMD3100 blocks both endogenous CXCL12 function and the effects of supplemental CXCL12 administration. (A) The asterisk (*) denotes a statistically significant difference (p < 0.01) versus Sham plus PBS; ^#p < 0.05, ^##p < 0.01 versus AS plus PBS; ^†p < 0.01 versus AS plus CXCL12; ^§p < 0.05 versus AS plus AMD3100; and for (B), *p < 0.01 versus Sham plus PBS; ^#p < 0.05 versus AS plus PBS; ^†p < 0.01 versus AS plus CXCL12. Sham and AS treated groups supplemented with bone marrow. (C) CD45^–ve population of GFP^+ve BMDSCs. *p < 0.05, **p < 0.01 versus Sham plus PBS; ^#p < 0.05 versus AS plus PBS; ^†p < 0.01 versus AS plus CXCL12. (D) CK^+ve population of GFP^+ve BMDSCs. *p < 0.01 versus Sham plus PBS; ^#p < 0.05 versus AS plus PBS; ^†p < 0.05 versus AS plus CXCL12. Data are presented as mean ± SEM.

Figure 3

Fibrosis Histology after trichrome (A) and Sirius red (B) staining of uterine tissue in controls and the five groups with supplemental bone marrow transplantation at 3 weeks after Asherman’s syndrome modeling. Original magnification, 40×. (A) Uninjured controls; (B) AS with PBS control treatment; (C) AS with CXCL12 treatment; (D) AS with AMD3100 treatment; (E) AS with CXCL12 plus AMD3100 treatment. Fibrosis is increased after injury and reduced by CXCL12. AMD3100 treatment increases fibrosis, likely by blocking both endogenous CXCL12 (D) and CXCL12 treatment (E).

Figure 4

Fibrosis Is Reduced by CXCL12 Fibrosis scorings of uterine tissues in the eight groups with supplemental bone marrow (BM) transplantation at 3 weeks after Asherman’s syndrome modeling. AS increased fibrosis, whereas CXCL12 reduced fibrosis in AS. AMD3100 reversed the effects of endogenous or supplemental CXCL12 administration. *p < 0.01 versus Sham plus BM plus PBS; ^#p < 0.05 versus AS plus BM plus PBS; ^†p < 0.01 versus AS plus BM plus CXCL12.

Figure 5

CXCL12 Restores Uterine Function Pregnancy outcomes among control and treated female groups were determined by mating with fertile wild-type males. Pregnancy rate is not significantly reduced in this mild AS model. However, blocking the ability of endogenous CXCL12 to spontaneously repair the uterus after injury by administration of AMD3100 did reduce pregnancy rate. Litter size and time to conceive are both significantly affected by injury in this model. CXCL12 treatment restored litter size and time to conceive. AMD3100 treatment blocked the effects of supplemental CXCL12. AMD3100 reduced pregnancy rate and litter size as well as increased time needed to conceive in this AS model. *p < 0.05, **p < 0.01 versus Control; ^#p < 0.05, ^##p < 0.001 versus AS plus PBS; ^†p < 0.05, ^††p < 0.01, and ^†††p < 0.001 versus AS plus CXCL12. Data are presented as mean ± SEM.