Bone Marrow-Derived Stem Cell (BMDSC) transplantation improves fertility in a murine model of Asherman's syndrome

Abstract

Asherman's Syndrome is characterized by intrauterine adhesions or fibrosis resulting as a consequence of damage to the basal layer of endometrium and is associated with infertility due to loss of normal endometrium. We have previously shown that bone marrow derived stem cells (BMDSCs) engraft the endometrium in mice and humans and Ischemia/reperfusion injury of uterus promoted BMDSCs migration to the endometrium; however, the role of BMDSCs in Asherman's syndrome has not been characterized. Here a murine model of Asherman's syndrome was created by traumatizing the uterus. We evaluate stem cell recruitment and pregnancy after BMDSCs transplantation in a model of Asherman's syndrome. In the Asheman's syndrome model, after BMDSC transplant, the Y chromosome bearing CD45-cells represented less than 0.1% of total endometrial cells. Twice the number of Y+CD45- cells was identified in the damaged uterus compared to the uninjured controls. There was no significant difference between the damaged and undamaged uterine horns in mice that received injury to a single horn. In the BMDSC transplant group, 9 of the 10 mice conceived, while only 3 of 10 in the non-transplanted group conceived (Chi-Square p = 0.0225); all mice in an uninjured control group conceived. The time to conception and mean litter size were not different between groups. Taken together, BMDSCs are recruited to endometrium in response to injury. Fertility improves after BMDSC transplant in Asherman's Syndrome mice, demonstrating a functional role for these cells in uterine repair. BMDSC transplantation is a potential novel treatment for Asherman's Syndrome and may also be useful to prevent Asherman's syndrome after uterine injury.

Figure 1. Histology of murine experimental Asherman's syndrome.

Three estrous cycles after uterus damage, the uterine horns were collected and stained with hematoxylin and eosin (H&E). (A) Fibrosis was observed in the damaged uterei. (B) No fibrosis was seen in uteri from control group. Original magnification, 400X.

Figure 2. Trichrome stain of fibrosis of murine experiment Asherman's syndrome.

The mice uterine horns were collected from the control group, experimental Asherman's syndrome group and BMT after uteri damage group. (A) No fibrosis was seen in uteri from the control group. (B) Fibrosis was observed in the Non- transplanted Asherman's group. (C) Fibrosis was also seen in BM-transplanted Asherman's group. Many animals in the treated group showed significantly decreased fibrosis. Original magnification, 400X.

Figure 3. Localized uterine damage recruits BMDSCs throughout the endometrium.

Immunofluorescence staining of Y chromosome (red) demonstrates male donor-derived BM cells localized in the endometrium of female recipients. Immunofluorescence staining of CD45 (green) distinguishes endometrial cells from leukocytes. Immunofluorescence staining of cytokeratin (yellow) indicates differentiated epithelial cells. DAPI is used to stain nuclei (blue). Top Panel: (A) Y chromosome signal in the testis shown as a positive control. (B) Y chromosome signal in male spleen tissue demonstrating expression of CD45 (pan leukocyte marker). (C) Y chromosome-positive, cytokeratin-positive, and CD45-negative cell, demonstrating a BM-derived endometrial epithelial cell.(D) Y chromosome-positive, cytokeratin-negative, and CD45-negative cell, demonstrating a BM-derived endometrial stromal cell in the endometrium (E) Y chromosome-positive, CD45-positive cell, demonstrating a transient leukocytes present in the endometrium. Lower Panel: Individual fluorescence images that result in merged figures 3 C and D from top panel. DAPI is used to stain nuclei (blue), Y chromosome FISH (red), immunofluorescence staining of cytokeratin (yellow) and CD45 (green) Original magnification, 400X.

Figure 4. Average litter size.

The average number of pups born is shown. (A) BM transplant Asherman'sgroup; (B) Non-BM transplant Asherman's group; (C) Control group. There is no significant difference between groups.