Towards the development of a bioengineered uterus: comparison of different protocols for rat uterus decellularization.

Uterus transplantation (UTx) may be the only possible curative treatment for absolute uterine factor infertility, which affects 1 in every 500 females of fertile age. We recently presented the 6-month results from the first clinical UTx trial, describing nine live-donor procedures. This routine involves complicated surgery and requires potentially harmful immune suppression to prevent rejection. However, tissue engineering applications using biomaterials and stem cells may replace the need for a live donor, and could prevent the required immunosuppressive treatment. To investigate the basic aspects of this, we developed a novel whole-uterus scaffold design for uterus tissue engineering experiments in the rat. Decellularization was achieved by perfusion of detergents and ionic solutions. The remaining matrix and its biochemical and mechanical properties were quantitatively compared from using three different protocols. The constructs were further compared with native uterus tissue composition. Perfusion with Triton X-100/dimethyl sulfoxide/H2O led to a compact, weaker scaffold that showed evidence of a compromised matrix organization. Sodium deoxycholate/dH2O perfusion gave rise to a porous scaffold that structurally and mechanically resembled native uterus better. An innovative combination of two proteomic analyses revealed higher fibronectin and versican content in these porous scaffolds, which may explain the improved scaffold organization. Together with other important protocol-dependent differences, our results can contribute to the development of improved decellularization protocols for assorted organs. Furthermore, our study shows the first available data on decellularized whole uterus, and creates new opportunities for numerous in vitro and in vivo whole-uterus tissue engineering applications.

Rejection of allogenic uterus transplant in the mouse: time-dependent and site-specific infiltration of leukocyte subtypes.

BACKGROUND: Animal models of uterus transplantation are being developed ahead of a possible treatment for absolute uterus infertility in women. Our knowledge of inflammatory cell involvement in acute rejection of a uterus transplant is limited; therefore, we examined the pattern of invasion of leukocyte subtypes into an allogeneic uterus transplant. METHODS: The uterus and its vasculature were removed from BALB/c mice and transplanted into C57Bl/6 recipient mice at a heterotopic position, with the native uterus left in situ. Both uteri were removed on post-operative day 2 (D2, n = 5), D5 (n = 5) and D10 (n = 6). Immunohistochemistry for neutrophilic granulocytes, macrophages, cytotoxic CD8(+) T-cells, CD4(+) T-helper cells and B-cells was performed and cell density was evaluated in both myometrium and endometrium. RESULTS: Neutrophil density was increased in graft versus native uteri at D5 and D10 in myometrium and D10 in endometrium, and in endometrium was higher in the D5 than D2 graft (all P < 0.05). Infiltration of macrophages occurred from D2 in myometrium and from D5 in endometrium (P < 0.05, graft versus native). Density of CD8(+) cytotoxic T-cells increased in the graft versus native uteri at D5 in both uterine layers and for the graft versus D2 density (P < 0.01). In contrast, CD4(+) T-helper cells increased only transiently in graft endometrium at D5 (P < 0.05). Overall CD19(+) B-cell density was low, with no time-dependent changes in graft myometrium or endometrium. CONCLUSIONS: Acute rejection of an allogeneic uterus transplant in the mouse involves influx of predominately neutrophils, macrophages, CD8(+) T-cells and CD4(+) T-cells between D2 and D5 post-operatively.

Rejection of the transplanted uterus is suppressed by cyclosporine A in a semi-allogeneic mouse model.

BACKGROUND: A mouse uterus transplantation model has previously been developed for studies of various aspects of uterine transplantation, which in the future may be used as treatment for uterine infertility. The aim of the study was to evaluate the effect of the immunosuppressant cyclosporine A (CyA) on the rejection of the allotransplanted uterus in the mouse. METHODS: C57BL/6 mice were recipients of uteri from F1 hybrids (C57BL/6 x CBA/ca). Transplanted mice received vehicle (control, n=5), 10 or 20 mg/kg/day of CyA (CyA10, n=5 and CyA20, n=5). Untreated F1 hybrids with syngeneic transplants (n=3) were negative controls. On day 10 post-transplantation, the grafted uteri were examined, and biopsies were taken for histology and quantification of T cells. RESULTS: Histology analysis revealed necrosis of the uterine transplants in controls and to a lesser extent in the CyA groups. Apoptosis and inflammation was prominent in grafts from the CyA10 group but suppressed in the CyA20 group. A similar increase of CD4+ cells was seen in all groups, whereas the number of CD8+ cells was higher (P < 0.05) in the two allogeneic groups receiving CyA compared with the allogeneic vehicle group. CONCLUSIONS: CyA treatment clearly delays the progress of rejection of grafted uteri but is insufficient to suppress T cell infiltration. Interestingly, the number of CD8+ cells was higher in groups receiving CyA, possibly reflecting a CyA-dependent depression of activation-induced cell death (AICD) of cytotoxic T cells.

Rejection patterns in allogeneic uterus transplantation in the mouse.

BACKGROUND: Transplantation of the uterus in the mouse has been developed as a model system for research towards human uterine transplantation. Previous studies in a mouse model have demonstrated that a syngeneic uterus transplant can give rise to normal offspring. The aim of this study was to characterize the time course of rejection in a fully allogeneic mouse uterus transplantation model. METHODS: Uteri of BALB/c mice were transplanted to a heterotopic position in C57BL/6 recipients, whose native uteri were left in situ. The blood flow of the uteri, their gross appearance and general histology and the density of T-lymphocytes were examined on postoperative days 2-28. RESULTS: Macroscopic signs of rejection were apparent from day 5. At the light microscopy level, minimal inflammatory changes were seen from day 5 and massive inflammation was seen from day 10 to day 15. At day 28, necrosis and fibrosis were seen. The density of T-lymphocytes (CD3(+)) was increased in the grafted uterus from day 2 in the myometrium and from day 5 in the endometrium. Blood flow in the grafted uteri was reduced from day 15. CONCLUSION: A murine model to study rejection of allogeneic uterus transplants was characterized. Signs of rejection were seen from day 2 to day 5 and severe rejection was seen from day 10 to day 15. The data will be useful in future studies on immunosuppressants in this model.

Short-term ischaemic storage of human uterine myometrium--basic studies towards uterine transplantation.

BACKGROUND: Transplantation of the uterus has been suggested as a possible future treatment of absolute uterine infertility. The tolerability of human uterine tissue to cold ischaemic storage was tested in the present study. METHODS: Small tissue samples of human uteri were subjected to cold (4 degrees C) ischaemia (6 and 24 h) in Ringer acetate (RIN), the intracellular-like University of Wisconsin solution (UW) or the extracellular-like Perfadex solution (PER). The ability of myometrial strips to contract, histology by light and electron microscopy as well as tissue concentrations of glutathione, ATP and protein were used as parameters to detect cold ischaemic injuries. RESULTS: Contractile ability and response to prostaglandin F(2alpha) (PGF(2alpha)) was better preserved after 6 h cold ischaemia in UW and PER in comparison with the other groups. Histological examination did not reveal any major changes after 6 and 24 h cold ischaemic storage in UW and PER solutions, while specimens stored in RIN for 24 h displayed degenerative changes on the electron microscopy level. UW and PER preserved ATP concentrations significantly better than RIN. Myometrium stored in UW contained more total glutathione but also a larger proportion of oxidized glutathione than specimens stored in RIN and PER. Protein concentrations did not change with storage time in any of the solutions. CONCLUSIONS: The results show that human uterine myometrial tissue is resistant towards cold ischaemia for at least 6 h if stored in UW and PER solutions.

Pregnancy in transplanted mouse uterus after long-term cold ischaemic preservation.

BACKGROUND: The aim of this study was to evaluate the viability of the transplanted murine uterus after cold ischaemic preservation. METHODS: Uteri of mice (6-8 weeks old) were isolated and kept at 4 degrees C in vitro for 24 or 48 h in 0.154 mol/l NaCl or University of Wisconsin (UW) solution. Viability was evaluated by assessment of morphology and contractility in vitro. Furthermore, uteri were transplanted by vascular anastomoses to syngeneic recipients after 24 or 48 h cold ischaemic preservation in UW solution and morphology, blood flow and capacity to implant transferred blastocysts were assessed 2 weeks later. RESULTS: Uteri that had been preserved for 24 h exhibited normal morphology but after 48 h preservation minimal degenerative changes were seen. Spontaneous contractions occurred in uteri after 24 h as well as 48 h cold ischaemic preservation and prostaglandin F(2alpha)-stimulated responses were preserved. Blood flow and morphology were normal 2 weeks after transplantation in uteri preserved for 24 h, while grafts preserved for 48 h had a decreased blood flow and morphology showed total necrosis of the transplants. Transplanted uteri that had been preserved for 24 h developed pregnancies (in five out of six animals) after embryo transfer, with offspring showing normal weight and growth trajectory. CONCLUSIONS: This study shows for the first time that the mouse uterus tolerates cold ischaemic preservation and that pregnancies can be carried in transplanted uteri that have been preserved for 24 h.

Heterotopic uterine transplantation by vascular anastomosis in the mouse.

A method of heterotopic uterine transplantation was developed in the mouse as a model system for studies of uterine function and transplant immunology of the uterus. The model involved transplantation of the right uterine horn and the cervix by vascular anastomosis to a donor animal with the intact native uterus remaining in situ. F1-hybrids of inbred C57BL/6 x CBA/ca (B6 CBAF1) mice of 6-8 weeks of age (n=42) were used. The specific pelvic vascular anatomy of these mice was first examined by intra-aortal injection of a two-component silicon-rubber curing agent. The surgery of the donor animal involved microsurgical isolation of the right uterine horn and the cervix, with preserved vascular supply from the aorta through the right uterine artery. After isolation of the uterine horn with vascular supply and venous drainage, including approximately 3 mm of the inferior vena cava and aorta, the organ was put on ice. The recipient animal was prepared by exposing and mobilizing the infrarenal part of the aorta and the vena cava. The grafted uterus was placed in the abdomen on the left side and the aorta and vena cava of the graft were anastomosed end-to-side to the aorta and vena cava of the recipient animal with 11-0 sutures. The total time for these procedures declined with time and was 125+/-4 min for the last 28 operations. Viability of the uterus was confirmed, several days later, by demonstrating a blood flow similar to that of the native uterus, and histology of the grafted uterus demonstrated normal morphology, including intact ultrastructure of the endothelial cells. The overall survival rate of the recipient animals increased with learning from approximately 40% in animals 1-21 to 71% in animals 22-42. The proportion of viable grafts, as judged by normal blood flow and histology among the surviving mice was 25% in animals 1-21 and 87% in animals 22-42. An undisturbed function of the transplanted uterus horn was finally demonstrated by its ability to implant inserted blastocysts and to carry pregnancy with fetal weight being similar to that of fetuses in the native uterus and controls. In conclusion, this is the first report of successful transplantation of the uterus with proven functionality in the mouse. The model should be useful for many aspects of research in uterine physiology and pathophysiology.

The antibody-neutralisation of PDGF, CSF-1, TGFb2,3, EGF and EGF-receptor in utero in pre-implantation mice.

The aim of this study was to determine whether specific growth factors, those shown to be involved using PCR and immunohistochemistry, are necessary for the in-vivo mechanisms of normal implantation in mice. The abdomen of pregnant female mice were opened surgically on day 4 to expose the uterine horns, which were microinjected with specific neutralising antibodies against PDGF, CSF-1, TGFb2,3, EGF and EGF-receptor. At autopsy on day 12, the numbers, positions and sizes of all implantation and resorption sites were recorded. Sham-operation controls were utilised to evaluate the implantation model. Normal female mice exhibited a mean of 6.24 implantation sites per uterine horn. Sham-operated mice exhibited a 30.8% reduction in implantation compared with normals, and saline-injected mice exhibited a 45.7% reduction. Antibody-injected horns were compared with horns injected with saline and horns injected with heat deactivated antibody. All neutralising antibodies tested resulted in significant reductions in the implantation rate and the size of the implantation site. These experiments confirm, in vivo, participation of the specific growth factors tested in the mechanisms of murine implantation, as alluded to previously by evidence from PCR in vitro stimulatory and immunohistochemical work.