Allogeneic uterus transplantation in a rhesus model: A short-term graft viability study.

Uterine factor infertility (UFI) is a major type of female infertility that is still considered untreatable. Available options include surrogacy and adoption. However, surrogacy is illegal in many nations including China [ 1 – 3 ]. On the other hand, due to the traditional culture in China, adoption is not as common as in it is in Western countries. Uterus transplantation (UTx) is a potential option for women with UFI who wish to have a child. Accumulative data from previous experimental studies have led to the clinical application of UTx. In 2014, almost 50 years after the initial concept was first proposed, the first live birth following UTx was reported [ 4 ]. The investigators suggest that UTx may be a feasible fertility-restoring intervention for women with UFI. Thus far, more than 60 UTx surgeries have been performed globally, and 18 offspring have been reported [ 5 ]. In recent decades, organ transplantation has been used in clinical practice. At the same time, assisted reproductive technology (ART) has progressed rapidly for live births after in vitro fertilization (IVF). The technology of human UTx benefits from the development of those technologies. Many clinical study teams have recently aimed for the clinical application of UTx on the basis of experimental data. However, many clinical and technical issues of UTx need to be resolved [ 6 ]. Since the parameters associated with UTx have not been standardized, basic data from animal experiments are needed for the further clinical application of UTx. Animal models are extensively used for the investigation of human disease and surgical procedures, such as endometriosis and organ transplantation. In recent years, animal studies on UTx have been performed with rats, rabbits, mice, dogs, swine, sheep, and nonhuman primates [ 7 ]. The outcomes of allogeneic UTx have been reported only in rats and sheep [ 8 , 9 ], and there are only a few cases of UTx in nonhuman primates [ 10 , 11 ]. UTx cases involving nonhuman primates would provide important preclinical guidance to resolve problems associated with UTx in clinical practice, since these animals are anatomically and physiologically similar to humans. The potential challenges of allogeneic UTx in nonhuman primates pertain to complicated surgical techniques, potential ischemia-reperfusion injury and rejection, and the delicate techniques used for vascular anastomosis. The evaluation criteria for the success of UTx include long-term survival of recipient animals and recovery of reproductive function. The anatomical pelvic structures of rhesus monkeys are almost identical to those of humans, and the experience of allogenic UTx in nonhuman primates can be used to investigate transplantation in humans. In this context, we aimed to evaluate the surgical technique for allogeneic UTx in a rhesus model.

In total, four rhesus monkeys were used in this study. The animals were designated as either a donor or recipient depending on their ABO blood type compatibility. The details of the UTx operation parameters of each animal are summarized in Table 1 . The average time of donor surgery was 235 minutes (min); the average time of uterine retrieval was 133 min; the average time of warm ischemia 1 * was 9 minutes; the average time of warm ischemia 2 * was 101.75 min; the average time of cold ischemia was 86.5 min; the average perfusion time was 23.75 min; the average vascular anastomosis time was 133 min; and the average blood loss was 20 milliliters (ml). *: warm ischemia 1 (donor): ischemia during implantation, from removal of the organ from ice until reperfusion; warm ischemia 2 (recipient): from cold storage to transplant vascular anastomosis completion and re-warming; cold ischemia: from organ retrieval to before vascular anastomosis. Dexamethasone (5 mg/day) was orally administered to all animals on the day before surgery as an induction treatment until two weeks post operation ( Table 2 ). MMF was administered orally 250 mg/day post operation, CyA was administered orally 25 mg/day post operation, and tacrolimus was administered orally 1 mg/day. No rejection reaction occurred in any rhesus monkey. DXM: dexamethasone; MMF: mycophenolate mofetil; CyA: cyclosporine a. The short-term outcome evaluation showed that the transplanted vessels had satisfactory intraoperative conditions in all rhesus monkeys. Doppler ultrasonography revealed that the blood flow of the external iliac artery was stable in all four animals at 1 and 4 weeks post operation. The average peak systolic velocity (PSV) of the bilateral external iliac artery blood flow was 10 cm/s and 10.3 cm/s at 1 and 4 weeks post operation, respectively. Ultrasonography showed that the flow resistance indexes (RIs) on the right side and left side were 0.35 and 0.38 at 4 weeks post operation, respectively. The average uterine length was 2.8cm. Subsequently, ultrasonography showed that the graft uterus in the animal had normal gross morphology. The menstruation resumed normally within 2 months after surgery, and two of the animals had regular menstruation periods. Premenstrual ultrasound revealed endometrial edema in the rhesus monkey. Histopathologically, there was no abnormality in the cervix ( Fig 5 ). Inflammatory findings with lymphocytes and fresh or remote hemorrhage could be found in the biopsy specimen of the cervix after surgery. This was a normal postoperative inflammatory response. A: Normal cervix, showing no marked inflammatory cell infiltration (original magnification ×20); B: Histopathological findings in a biopsy specimen of the cervix at 5 days after surgery, showing inflammatory findings consisting of lymphocytes and fresh bleeding (blue arrow, original magnification ×20); C: Histopathological findings in the biopsy specimen of the cervix at 25 days after surgery, showing inflammatory findings with lymphocytes and remote hemorrhage (yellow arrow, original magnification ×20).

This report shows the safety and feasibility of allogeneic UTx technology in the rhesus model. No organ or vessel injury was observed in any of the donors or recipients. This is the first report to evaluate the functional recovery of a rhesus model after allogeneic UTx. We hope this research will provide a scientific foundation for future studies in primates and provide preclinical insights for human UTx.

Four female rhesus monkeys with regular menstrual cycles were purchased from an animal experimental center. The mean body weight of all the rhesus monkeys was 5.325 kg, while no pregnancy was noted preoperatively. To reduce postoperative immune rejection, the animals were selected as either donors or recipients (n = 4) on the basis of ABO blood type compatibility. The general condition of each rhesus monkey was examined 1–2 weeks before surgery. After the experimental animal was verified to be in good condition post surgery, she was housed for use in further research, such as embryo transplantation. All the rhesus monkeys were acclimatized to their new surroundings for at least 7 days to reduce possible pressure. All rhesus monkeys were housed in separate cages with adequate food and water. The room temperature varied from 19°C to 25°C, with a relative humidity of 40–60% and a 12-hrs light/dark cycle. Animal health and behavior were monitored twice daily. The animal was euthanized within 4 hrs if any of the following conditions occurred: weight loss, pain that could not be effectively controlled, excessive tumor growth or ascites production, persistent self-harm behavior, systemic hair loss caused by disease, long-term diarrhea that could not be treated due to experimental factors, persistent fatigue accompanied by rough fur, an arched back, an enlarged abdomen, inability to walk, severe anemia, jaundice, abnormal central nervous system response, uncontrollable bleeding, abnormal urination, disorders affecting eating and drinking, end-stage infectious diseases, severe low temperature, and obvious functional impairment. In this study, no animals died before meeting the criteria for euthanasia. All personnel participating in the animal experiment received animal experiment training and obtained the qualification certificate of animal experiment practitioners. The study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council and approved by the Ethics Committee of Peking University Third Hospital (No. LA2016317). The blood group compatibility test method included bloodgrouping and crossmatching. The reagents were added in the following order: 1) human anti-A erythrocyte suspension + serum to be tested; 2) human anti-B erythrocyte suspension + serum to be tested; 3) human anti-B serum + erythrocyte suspension to be measured; and 4) human anti-A serum + erythrocyte suspension to be measured. The major and minor crossmatch tests were included in the compatibility test [ 12 ]. The major crossmatch tests for alloantibodies in the recipient’s plasma against donor red cells and the minor crossmatch tests for alloantibodies in the donor’s plasma against the recipient’s red blood cells. The presence of agglutination on either test indicated that the recipient was not compatible with the donor’s red blood cells (major) or with the donor’s plasma (minor) [ 13 ]. The hair of each rhesus monkey was clipped on the lower abdominal region, and food was withdrawn 24 hrs before the day of surgery. After being sedated, the animal was injected with 3% pentobarbital sodium (Shanghai Harring Biotechnology Co., Ltd). The abdomen was sterilized, the animal was placed in the supine position, and vein puncture catheterization was performed. A tracheal tube was inserted with a positive airway pressure breathing machine connected, and anesthesia was maintained with 3% pentobarbital sodium (Shanghai haring Biotechnology Co., Ltd.). All animals received continuous intravenous infusion to compensate for the loss of fluid during surgery. For prophylaxis antibiotics were administered from the initiation of the operation until one week after surgery. First, laparotomy through a midline incision was performed in the donor animal. The uterine artery, adjacent internal iliac artery, and ovarian uterine veins were dissected separately ( Fig 1A ). Then, the vaginal artery and vessels surrounding the uterus were dissected separately ( Fig 1B ). The ovaries and oviducts of donors were preserved in situ ( Fig 1A ). The isolated uterine tissue was stored at 4°C in organ preservation solutions and perfused with 300–350 milliliters (ml) of cold histidine-tryptophan-ketoglutarate solution (HTK®; Custodiol®, Nordmedica, Gentofte, Denmark) via a perfusion catheter (22-G intravenous needle) in the uterine artery ( Fig 2A ). The uterus was maintained under these conditions until the lavage outflow became clear ( Fig 2B ). During recipient surgery, the uterine graft was brought into the operative field, and the vagina of the graft was anastomosed with the vaginal vault of the recipient to fix the uterus in the pelvis ( Fig 3A ). Before vascular anastomoses, heparin (100 IU) was administered intravenously. The vascular clamps were placed on both ends of the isolated segments of the external iliac artery. Then, the bilateral internal iliac artery was anastomosed end-to-side with the iliac externa of the arteries, and bilateral ovarian uterine veins were anastomosed end-to-side with the vena iliac externa using continuous sutures (7–0 or 8–0 Prolene®) ( Fig 3B ). The clamps were released after the conclusion of all vascular anastomoses, and homeostasis was evaluated. The severe leakage was controlled by placing additional sutures. Anastomotic patency and blood flow were assessed by the pulsations of the uterine arteries and the color of the uterus. The vaginal cuff was then reattached to the vagina by interrupted sutures (1–0 polyglactin). A: a-ovarian uterine veins; b-uterine artery. B: a-cervical; b-uterine artery; c-internal iliac artery; d-external iliac artery; e-external iliac veins. A: a-cold histidine-tryptophan-ketoglutarate solution via a perfusion catheter (22-G intravenous needle)-perfused uterine artery. B: Lavage outflow clear and pale uterus. A: a-Transplantation of the internal iliac artery end-to-side anastomosis with the external iliac artery from the recipient, b-Transplantation of the ovarian uterine veins end-to-side anastomosis with the external iliac veins from the recipient. B: The grafting rapid vascular filling after anastomosis. The size and blood flow of the transplanted uterus were determined by transabdominal ultrasonography after closing the abdominal incision by interrupted sutures (1–0 polyglactin) ( Fig 4 ). After the surgical procedure, animals were separately accommodated in a single cage. The general condition of each rhesus monkey in terms of the following aspects was evaluated daily: appetite, bowel movements, vomiting, urination, attitude, and laparotomy wound site. To monitor for potential rejection after surgery, the size of the transplanted uterus and blood flow in the transplanted uterine artery were determined by transabdominal ultrasonography under anesthesia in the first and fourth weeks after the operation. Cervical tissue biopsy was performed before the operation, 5 days after the operation, and 25 days after the operation. Dexamethasone (DXM) was administered to all animals as an induction treatment. Mycophenolate mofetil (MMF), cyclosporine a (CyA), and tacrolimus were administered orally.

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