Endometrial cells contribute to preexisting endometriosis lesions in a mouse model of retrograde menstruation†

Abstract

Endometriosis is characterized by extrauterine growth of endometrial tissue accompanied by adverse clinical manifestations including chronic pelvic pain and infertility. Retrograde menstruation, the efflux of endometrium into the peritoneal cavity during menstruation, is believed to contribute to implantation of endometrial tissue and formation of endometriotic lesions at ectopic sites. While it is established through various rodent and nonhuman primate models that endometrial tissue fragments, as well as nondissociated stroma and glands, are capable of seeding endometriosis in a manner mimicking retrograde menstruation, the ability of single endometrial cells to participate in endometriotic processes has not been evaluated due to their failure to establish macroscopic endometriosis. We designed a model by which this capacity can be assessed by examining the integration of individual uterine cells into existing endometriosis lesions in mice. Endometriosis was induced in C57BL/6J female mice followed by intraperitoneal injection of GFP-labeled single uterine cells. We found that freshly introduced uterine cells can successfully integrate and contribute to various cell populations within the lesion. Strikingly, these cells also appeared to contribute to neo-angiogenesis and inflammatory processes within the lesion, which are commonly thought of as host-driven phenomena. Our findings underscore the potential of individual uterine cells to continuously expand lesions and participate in the progression of endometriosis. This model of retrograde menstruation may therefore be used to study processes involved in the pathophysiology of endometriosis.

Keywords: endometriosis; endometrium; retrograde menstruation.

Figure 1.

Mouse model of retrograde menstruation. (A) One-week post EMI, female mice were injected with either PBS or single-cell suspension containing 1 × 10⁶ GFP-labeled viable uterine cells. (B) Flow cytometric analysis of GFP+ uterine cells integrated in the endometriosis lesion 4 weeks post EMI. Representative scatter plots from PBS (n = 5) and uterine cell injection (n = 14). Values in boxes denote percentages out of the live cell population.

Figure 2.

Localization of uterine cells integrated into the endometriotic lesion. Representative immunohistochemical section of an endometriotic lesion demonstrating brown-stained GFP+ uterine cells found in the lesion stroma as well as integrated into blood vessels wall in the lesion (arrowheads) (n = 8, right). Representative section of a lesion from PBS-injected mice (n = 4, left). Lower panel: higher magnification of boxed areas.

Figure 3.

Enrichment of hematopoietic cells derived from cell injection in the endometriosis lesion. Flow cytometric analysis of the percentage of GFP+ cells expressing the pan-hematopoietic marker CD45. Representative plots from PBS (n = 5) and uterine cell injection (n = 14). Values in boxes denote percentages out of the live cell population.

Figure 4.

Profile of uterine cells incorporated into growing mouse endometriosis lesions. Immunofluorescent micrographs of endometriosis lesions 4 weeks post EMI. Representative sections from control (n = 4) and experimental (n = 8) lesions from different mice are shown. Co-staining of GFP-positive uterine cells integrated into a growing endometriosis lesion (green) with (A) CD31, (B) CD45 (section depicting an inflammatory cluster within the lesion), (C) vimentin, (D) PCNA, and (E) CK8 (red). Sections were counterstained with DAPI showing nuclei (blue). Arrowheads point to GFP+ integrated uterine cells. Right column: higher magnification images of dashed areas. Scale bar = 50 μm. (F) Quantification of the percentage of cells double-positive for GFP and CD31 (n = 7 lesions), vimentin (n = 7 lesions), or PCNA (n = 7 lesions).