First Successful Delivery after Uterus Transplantation in MHC-Defined Cynomolgus Macaques

Abstract

Delivery following uterus transplantation (UTx)-an approach for treating uterine factor infertility-has not been reported in nonhuman primate models. Here, six female major histocompatibility complex (MHC)-defined cynomolgus macaques that underwent allogeneic UTx were evaluated. Antithymocyte globulin and rituximab were administered to induce immunosuppression and a triple maintenance regimen was used. Menstruation resumed in all animals with long-term survival, except one, which was euthanized due to infusion associated adverse reaction to antithymocyte globulin. Donor-specific antibodies (DSA) were detected in cases 2, 4, and 5, while humoral rejection occurred in cases 4 and 5. Post-transplant lymphoproliferative disorder (PTLD) developed in cases 2 and 3. Pregnancy was attempted in cases 1, 2, and 3 but was achieved only in case 2, which had haploidentical donor and recipient MHCs. Pregnancy was achieved in case 2 after recovery from graft rejection coincident with DSA and PTLD. A cesarean section was performed at full-term. This is the first report of a successful livebirth following allogeneic UTx in nonhuman primates, although the delivery was achieved via UTx between a pair carrying haploidentical MHCs. Experimental data from nonhuman primates may provide important scientific knowledge needed to resolve unsolved clinical issues in UTx.

Keywords: allogeneic uterus transplantation; cynomolgus macaque; donor specific antibody; humoral rejection; post-transplant lymphoproliferative disorder; uterine factor infertility; uterine transplantation; uterus transplantation.

Figure 1

Donor and recipient surgery. (A) Schema of abdominal vessels dissected during donor surgery. Cross-clamping was performed by clamping the supraceliac aorta under the diaphragm and dividing the inferior vena cava (IVC) in the pleural space. A perfusion catheter was placed in the unilateral external iliac artery. (B) Dissection of the abdominal vessels and tissues surrounding the uterus before procurement of the uterus via donor surgery. Infrarenal aorta/IVC above the left renal vein, iliac and uterine vessels, and ovarian veins were dissected. Subsequently en bloc procurement of the uterus with these vessels and proximal vagina was performed. (C) Vascular anastomosis in recipient surgery. End-to-side venous anastomosis between the IVC of the donor and that of the recipient (white triangle) and between the abdominal aorta of the donor and the abdominal aorta of the recipient (yellow triangle). (D) Transplanted uterus. Following reperfusion, the color of the transplanted uterus changed from white to red without uterine congestion. (E) Intraoperative indocyanine green (ICG) fluorescent angiography in the transplanted uterus. ICG fluorescent angiography showed that there was sufficient blood flow to the transplanted uterus immediately following reperfusion (before (upper panel) and after (lower panel) injection of ICG).

Figure 2

Histologic variables of cellular and humoral rejection in cynomolgus macaques following uterus transplantation. (A) Normal squamous epithelium of the cervix <E0>. (B) Focal vacuolar alteration of the basal cell layer with mild junctional lymphocytic infiltrates <E1>. (C) Desquamative change of squamous epithelium. A small amount of residual epithelium is highlighted by a blue arrow <E3>. (D) Blood vessels and fibromuscular stroma with no significant inflammation <S0 and A0>. (E) Perivascular inflammation without endotheliitis <S1>. (F) Inflammation with endotheliitis in addition to perivascular inflammation, subendothelial lymphocytic infiltration is noted (yellow arrowheads). Venous endothelium demonstrates desquamative change and nuclear swelling <S2 or S3>. (G) Fibrin thrombi (*) in the small vessels without tissue necrosis <A1>. (H) Fibrin thrombi (*) in the small vessels surrounded by coagulative necrosis <A2>. (I) Immunohistochemistry for C4d using the same specimen from (H). Positive C4d immunostaining was seen along subendothelial spaces and in venous lumen. (A–H) H&E stain, bar = 100 µm. (I) Immunohistochemistry for C4d, bar = 100 µm.

Figure 3

Intracytoplasmic sperm injection and embryo transfer to the grafted uterus. (A) Blastocyst-stage embryo for embryo transfer is hatching from zona pellucida. (B) Follicular development of ovarian preovulatory follicle (yellow triangles) was observed via laparoscopy. (C) Ovulation (white arrow) was confirmed using laparoscopy. (D) Embryo intrafallopian transfer was performed after confirmation of ovulation. Embryos were inserted into the fallopian tube through a catheter. Fm, fimbria of oviduct.

Figure 4

Change of trough levels of tacrolimus in case 1 and 2 (A) and case 3 to 6 (B). In case 2, TAC was withdrawn from POD 191 to 220 due to PTLD development, resulting in occasional low trough levels during the period (arrow).

Figure 5

Rejection episodes and immunosuppressive treatment. Results of histopathological examination, immunosuppressive treatment, and cause of sacrifice are shown. Horizontal bars represent the length of animal survival following uterus transplantation. Results of uterine biopsy are depicted under each horizontal bar (NR, no rejection; BC, borderline change; CR, cellular rejection; HR, humoral rejection; LG, low-grade; HG, high-grade). Immunosuppressive treatments are depicted on each horizontal bar (R: rituximab, open triangle; A: antithymocyte globulin, closed triangle; P: steroid pulse treatment, closed arrow; MMF, mycophenolate mofetil; AZ, azathioprine; TAC, tacrolimus) The causes of sacrifice are shown at the end of the survival bar. The presence, including period of serum donor-specific antibody (DSA) (gray fill) and pregnancy (black fill) are indicated within the bars. Case 3 suddenly died due to PTLD and hyperpotassemia although the clinical course seemed to be satisfactory with no rejection and recovery of periodic menstruation. Case 6 was euthanized after closing the abdomen due to infusion associated reaction which occurred after administering antithymocyte globulin during surgery. PTLD, Post-transplant lymphoproliferative disorder; C, caesarian section.

Figure 6

Macroscopic findings from autopsy of cynomolgus macaques with humoral rejection. (A) ICG fluorescent angiography of the transplanted uterus in autopsy of case 4. Enhancement of the grafted uterus (yellow triangles) was absent in the image (before (upper panel) and after (lower panel) injection of ICG). (B) Macroscopic findings of the abdomen in the autopsy of case 5, showing a whitish swollen transplanted uterus (yellow triangles) with severe adhesion to the surrounding tissues (left panel). The grafted aorta was along the long axis and intravascular thrombi (white arrow) were found in the anastomosis site of aortas (yellow triangles) to the grafted common iliac arteries (right panel). AD, aorta of the donor; AR, aorta of the recipient; Bld, bladder; OV, ovary; Ut, uterus.

Figure 7

Development of PTLD with long-term graft survival after uterus transplantation in cynomolgus macaques. (A) Case 2 developed PTLD tumors in the anterior thorax and axilla on postoperative day (POD) 180 (left panel). The tumors disappeared completely one month following treatment due to repetitive administering of rituximab and cessation of MMF and TAC (right panel). (B) Biopsy specimen diagnosed as PTLD. Loupe image of needle biopsy specimens (H&E stain, bar = 4 mm); (upper left panel). Proliferation of atypical large lymphocytic cells are observed (H&E stain, bar = 100 μm); (upper right panel). Large atypical cells are positive for CD20; bar = 100 μm (lower left panel). Large atypical cells are positive for EBER in situ hybridization, bar = 100 μm (lower right panel). (C) Autopsy of case 3 showing tumors (yellow triangles) in the pleural cavity and mediastinum, diagnosed as PTLD.

Figure 8

Changes in peripheral lymphocyte counts. Peripheral lymphocyte (A), CD3+ (B), CD20+ (C) and NK cell (D) counts decreased after induction treatment with antithymocyte globulin (ATG) and rituximab (white arrow). CD3+ and CD20+ counts gradually increased following induction treatment, and then increased remarkably with rejection in some cases (black arrow). In case 2, lymphocyte and CD3+ cell counts elevated on POD 200 with low-grade cellular rejection caused by cessation of TAC and MMF due to treatment for PTLD, while CD20+ was suppressed due to administering of rituximab for PTLD.

Figure 9

Antibody responses against donor cells in case 1 (A), case 2 (B), case 3 (C), case 4 (D), and case 5 (E). FITC-labeled anti-monkey IgG antibodies were used as secondary antibodies. After gating at CD3-positive lymphocytes and CD20-positive lymphocytes, we made FITC-histograms to analyze IgG specific for each lymphocyte. Histograms of MHC-I (left) and MHC-II (right) exhibiting IgG antibodies specific for donor cells gated by CD3+ cells and CD20+ cells, respectively. In case 2, DSA specific for MHC class II was detected on POD 38, in addition to which DSA specific for MHC class I was detected on POD 48 (data not shown). Higher levels of DSA specific for MHC class I was detected on POD 96. Thereafter, the titer of MHC-class I-specific DSA gradually decreased and vanished on POD 180 when PTLD occurred, while low levels of MHC class II-specific DSA remained (B; upper panel). After PTLD was treated, MHC class I and II-specific DSA were not detected until delivery during the period from POD 212 to POD 1182 (B; lower panel). In case 4, in which a uterus with full-mismatched MHCs was transplanted, DSA was detected on POD 65 and similar levels of DSA was also detected on POD 83 and 101 (D). Low levels of MHC class I and II-specific DSA were detected in case 5, in which a uterus with mismatched MHC, except for one locus of MHC class IB, was transplanted (E). DSA was not detected in cases 1 and 3, in which multiple loci of MHC were matched between donor and recipient. (A,C).

Figure 10

Mixed Lymphocyte Reaction (MLR) stimulation indexes in pre- and post-transplantation. The MLR stimulation index decreased following induction treatment (white arrow). This index increased in conjunction with rejection in cases 1, 4, and 5. The index on POD 1363 in case 1 and on POD 1184 (on delivery) in case 2 were 2.10 and 1.33, indicating that index levels were low (data not shown).

Figure 11

First pregnancy and delivery after allogeneic uterus transplantation in a cynomolgus macaque. (A) Fetal heartbeat as per Doppler ultrasonography. Fetal viability was confirmed by transabdominal ultrasonography in case 2, with a crown-rump length of 11.7 mm and fetal cardiac activity. (B) Intraoperative findings of the cesarean section. Transverse incision of the lower part of uterus was performed.

Figure 12

Placenta accreta in transplanted uterus. (A) Placenta accreta was observed intraoperatively during cesarean section. Placenta was firmly adhered to the fundus of transplanted uterus (yellow triangles). (B) Resected transplanted uterus. Rests of partial placenta was observed in the fundus of the transplanted uterus (white triangles). (C) Delivery of the offspring from the transplanted uterus. (D) First newborn offspring after allogeneic uterus transplantation in a cynomolgus macaque. The body weight of the offspring was 358.4 g at delivery, indicating satisfactory growth during the pregnancy.