Long-Term Outcome and Rejection After Allogeneic Uterus Transplantation in Cynomolgus Macaques

Abstract

Uterus transplantation (UTx) is an option for women with uterine factor infertility to have a child, but is still in the experimental stage. Therefore, allogeneic animal models of UTx are required for resolution of clinical issues. In this study, long-term outcomes were evaluated in four recipients (cases 1-4) after allogeneic UTx in cynomolgus macaques. Immunosuppression with antithymocyte globulin induction and a triple maintenance regimen was used. Postoperative ultrasonography and biopsy of the transplanted uterus and immunoserological examinations were performed. All four recipients survived for >3 months after surgery, but continuous menstruation did not resume, although temporary menstruation occurred (cases 1 and 2). All animals were euthanized due to irreversible rejection and no uterine blood flow (cases 1, 2 and 4) and post-transplant lymphoproliferative disorder (case 3). Donor-specific antibodies against MHC class I and II were detected in cases 1, 2 and 4, but not in case 3. Peripheral lymphocyte counts tended to elevate for CD3⁺, CD20⁺ and NK cells in conjunction with uterine rejection, and all animals had elevated stimulation indexes of mixed lymphocyte reaction after surgery. Establishment of allogeneic UTx in cynomolgus macaque requires further exploration of immunosuppression, but the clinicopathological features of uterine rejection are useful for development of human UTx.

Keywords: allogeneic uterus transplantation; cynomolgus macaque; rejection; uterine factor infertility; uterine transplantation; uterus transplantation.

Figure 1

Changes of trough level in immunosuppressive treatment. (A) Cyclosporine concentrations in cases 1 and 2. (B) Tacrolimus concentrations in cases 3 and 4.

Figure 2

Macroscopic and histopathological findings in case 1. (A) Histopathological findings of a biopsy specimen from the uterine cervix on postoperative day (POD) 70. Severe stromal inflammation and capillary endotheliitis including lymphocytes and eosinophils were observed. Lymphocytes also infiltrated into squamous epithelium and resulted in liquefaction degeneration (white arrow). Necrotic change was also seen on the left side (within dotted line). These findings suggest severe rejection. Hematoxylin and eosin (H&E) stain, 200 × bar = 200 µm. (B) Histopathological findings of a biopsy specimen from the uterine body in second-look surgery on POD 133. In the necrotic and hyalinizing areas of the uterus, CD8-rich lymphocyte infiltration is seen. The white arrow shows a capillary vessel with an endothelium detached by attack of CD8 T-cells. CD8 IHC (immunohistochemistry), 400 × bar = 100 µm. (C) Macroscopic findings in the pelvis in autopsy on POD 196. A pale uterus with atrophy (yellow triangles) and left hydrosalpinx (white arrow) were observed. Ut, Uterus; Ov, Ovary; Od, Oviduct. (D) Intraoperative indocyanine green (ICG) fluorescence imaging of the transplanted uterus in autopsy. Enhancement of the uterus (yellow triangles) was absent in imaging. Ut, Uterus. (E) Histopathological findings of the grafted vessel in autopsy. Fibrotic occlusion with hemosiderin deposition is seen at the grafted vessel. Elastica van Gieson (EVG) stain, 100 × bar = 500 µm. (F) Histopathological findings of the donor’s left oviduct in autopsy. The donor’s grafted oviduct showed dilation and lymphocyte infiltration and vacuolar alteration (white arrow), which suggests mild rejection. Bar = 100 µm.

Figure 3

Macroscopic and histopathological findings in case 2. (A) Macroscopic findings in the pelvis in second-look surgery on POD 35. A markedly swollen uterus was observed. (B) Histopathological findings of the uterine myometrium in second-look surgery on POD 35. A small vessel in the myometrial interstitium showed severe endotheliitis. Due to lymphocytes infiltration, the endothelium is desquamated (white arrow). H&E stain, 200 × bar = 200 µm. (C) Transabdominal ultrasonography of the long axis of the uterine body on POD 97. An enlarged uterine body (33.5 × 23.2 mm: long axis × anteroposterior diameter) with a pooled abscess (yellow triangles) in the uterine cavity was found. (D) Macroscopic findings in the pelvis in autopsy on POD 126. A whitish atrophic uterus (yellow triangles) and bilateral hydrosalpinx (white arrow) were observed. Ut, uterus. (E) ICG fluorescence imaging of the transplanted uterus in autopsy. Enhancement of the grafted uterus (yellow triangles) was absent in imaging. Ut, uterus. (F) Histopathological findings of the removed uterus in autopsy on POD 196. The uterus had no endometrium. Neutrophilic exudate is seen, which suggests infection (*). On the bottom half, stromal tissue shows lymphocyte infiltration with capillary endotheliitis, indicating continuous rejection. Bar = 1mm.

Figure 4

Macroscopic and histopathological findings in case 3. (A,B) Histopathological findings of a biopsy specimen from the uterine cervix on POD 83. Lymphocytes infiltrated the epithelium and stroma with mild vacuolar alteration (white arrow). A Civatte body was seen (yellow triangle). H&E stain, 200×. (A). Most of these lymphocytes were positive for CD8. CD8-IHC, 200×. (B) Bar = 200 µm. (C) The right upper eyelid of this animal was swollen. (D) Macroscopic findings in the pelvis in autopsy on POD 140. A reddish uterus of normal size was observed in the pelvis and a 4 cm tumor was present at the site of the abdominal aorta (yellow triangles). Ut, uterus. (E,F) Histopathological findings of a resected nodular tumor, showing monomorphic large-sized lymphoid cells with abundant nucleoli. H&E stain, 200x. (E). Bar = 100 µm. These atypical cells were positive for CD20 and EBER-ISH (window). (F). Bar = 200 µm.

Figure 5

Macroscopic and histopathological findings in case 4. (A) Histopathological findings of a uterine tissue biopsy on POD 41. Perivascular lymphocyte infiltration and endotheliitis (white arrow) with perivascular fibrosis were clearly observed. Bar = 200 µm. (B) Macroscopic findings of the abdomen in autopsy on POD 104, showing a whitish swollen transplanted uterus. Severe adhesion at a site surrounding vascular anastomosis was present (yellow angle). The right adnexa was detected, but the left ovary and oviduct were not detected due to severe adhesion. Ov, ovary; Ut, uterus; F, fimbria; Bld, bladder. (C) The site of vascular anastomosis between aortas of recipient and donor. The aorta of the recipient was dissected and observation of the anastomotic site (yellow circle) showed that almost all of the lumen of the grafted aorta of the donor was stenosed, resulting in a pin hole (white triangle). Ao, aorta; GAo, grafted aorta; Ut, uterus. (D) Removed grafted (donor) and recipient vessels. The grafted aorta was along the long axis and intravascular thrombi were found from the anastomotic site (white arrow) to the grafted common iliac arteries (yellow triangles). Ao, aorta; Gao, grafted aorta; IVC, inferior vena cava; CIA, common iliac artery; CIV, common iliac vein; Ut, uterus. (E) Histopathological findings in the cervix of the removed uterus. Severe endotheliitis and epithelial desquamation (yellow arrows) were seen in the cervix. Bar = 500 µm. (F) Histopathological findings of the removed grafted aorta. Lymphocytes did not attack the endothelium (*) of the aorta directly; however, its feeding vessels in the vascular adventitia showed severe endotheliitis and obstruction (yellow triangles). Bar = 500 µm. VL, vascular lumen.

Figure 6

Changes in peripheral lymphocyte counts. Lymphocyte counts decreased after administration of antithymocyte globulin (ATG). The nadir lymphocyte counts in cases 3 and 4 were prolonged by differences in ATG doses, in comparison with counts in cases 1 and 2.

Figure 7

Changes in peripheral cell counts and ratio. (A) CD3⁺; (B) CD20⁺ and (C) NK cell⁺ counts decreased immediately after induction treatment, and then gradually increased. (D) The CD4⁺/CD8⁺ ratio showed a tendency to decrease.

Figure 8

Mixed lymphocyte reaction (MLR) stimulation indexes pre- and post-transplantation. (A) Case 1. (B) Case 2. (C) Case 3. (D) Case 4.

Figure 9

Changes of donor-specific antibodies (DSAs). Pre- and post-transplantation DSAs were produced against MHC class I and II in all animals except for case 3. The titers of DSAs increased thereafter.