A decellularized uterine scaffold (DUS) prepared from rats permits recellularization and regeneration of uterine tissues when placed onto a partially excised uterus and supports pregnancy in a fashion comparable to the intact uterus. The underlying extracellular matrix (ECM) together with an acellular, perfusable vascular architecture preserved in DUS is thought to be responsible for appropriate regeneration of the uterus. To investigate this concept, we examined the effect of the orientation of the DUS-preserving ECM and the vascular architecture on uterine regeneration through placement of a DUS onto a partially defective uterine area in the reversed orientation such that the luminal face of the DUS was outside and the serosal face was inside. We characterized the tissue structure and function of the regenerated uterus, comparing the outcome to that when the DUS was placed in the correct orientation. Histological analysis revealed that aberrant structures including ectopic location of glands and an abnormal lining of smooth muscle layers were observed significantly more frequently in the reversed group than in the correct group (70% vs. 30%, P < 0.05). Despite the changes in tissue topology, the uteri regenerated with an incorrectly oriented DUS could achieve pregnancy in a way similar to uteri regenerated with a correctly oriented DUS. These results suggest that DUS-driven ECM orientation determines the regenerated uterus structure. Using DUS in the correct orientation is preferable when clinically applied. The disoriented DUS may deteriorate the tissue topology leading to structural disease of the uterus even though the fertility potential is not immediately affected.

Partial or whole regeneration of the uterine endometrium using extracellular matrix (ECM)-based scaffolds is a therapeutic strategy for uterine infertility due to functional and/or structural endometrial defects. Here, we examined whether the entire endometrium can be regenerated circumferentially using an acellular ECM scaffold (decellularized endometrial scaffold, DES) prepared from rat endometrium. We placed a silicone tube alone to prevent adhesions or a DES loaded with a silicone tube into a recipient uterus in which the endometrium had been surgically removed circumferentially. Histological and immunofluorescent analyses of the uteri one month after tube placement revealed more abundant regenerated endometrial stroma in the uterine horns treated with tube-loaded DES compared to those treated with a tube alone. Luminal and glandular epithelia, however, were not fully recapitulated. These results suggest that DES can enhance the regeneration of endometrial stroma but additional intervention(s) are needed to induce epithelization. Furthermore, the prevention of adhesions alone allowed the endometrial stroma to regenerate circumferentially even without a DES, but to a lesser degree than that with a DES. The use of a DES together with the prevention of adhesions may be beneficial for efficient endometrial regeneration in the uterus that is largely deficient of endometrium.

To clarify whether a mediator complex subunit 12 (MED12) gain-of-function mutation induces leiomyoma cell properties in human uterine smooth muscle cells (USMCs). Experimental study. Academic research laboratory. Women undergoing hysterectomy for leiomyoma. CRISPR/Cas9-mediated genome editing to introduce an MED12 gain-of-function mutation (G44D) into human USMCs. Cell proliferation, collagen production, and in vivo tumorigenicity of USMCs with vs. without the MED12 mutation. Uterine smooth muscle cells isolated from the uterine myometrium of a 44-year-old patient were subjected to lentiviral vector-mediated gene transduction of the fluorescent protein Venus, followed by long-term passage. Uterine smooth muscle cells with a normal female karyotype, high cell proliferative activity, and Venus expression, but without stem/progenitor cell populations, were obtained and designated as USMC44. Using CRISPR/Cas9-mediated genome editing, mtUSMC44 (MED12, 131G>A, p.G44D) and mock USMC44 without MED12 mutation (wtUSMC44) were established from USMC44. wtUSMC44 and mtUSMC44 showed similar cell proliferation activity, even in the presence of estradiol and progesterone (EP) together with transforming growth factor-beta 3 (TGFB3). In addition, wtUSMC44 and mtUSMC44 generated similar tiny smooth muscle-like tissue constructs when xenotransplanted beneath the kidney capsule in immunodeficient mice treated with EP alone or TGFB3. In contrast, mtUSMC44 produced more collagen type I than wtUSMC in vitro, and this production was likely enhanced by EP and TGFB3. The results suggest that the MED12 gain-of-function mutation is involved in collagen production. Although approximately 70% of leiomyomas have MED12 mutations, additional factors and/or events other than MED12 and/or myometrial stem/progenitor cells may be required for fully inducing leiomyoma cell properties, including transformation, in USMCs.