Journal of

Clinical Medicine
ArticleLong-Term Outcome and Rejection After Allogeneic
Uterus Transplantation in Cynomolgus Macaques
Iori Kisu 1, * , Hirohito Ishigaki 2 , Katsura Emoto 3 , Yojiro Kato 4 , Yohei Yamada 5 ,Kentaro Matsubara 6 , Hideaki Obara 6 , Yohei Masugi 3 , Yusuke Matoba 1 , Masataka Adachi 1 ,Kouji Banno 1 , Yoko Saiki 7 , Iori Itagaki 8,9 , Ikuo Kawamoto 8 , Chizuru Iwatani 8 ,Takahiro Nakagawa 8 , Hideaki Tsuchiya 8 , Takako Sasamura 2 , Hiroyuki Urano 10 ,Masatsugu Ema 8 , Kazumasa Ogasawara 2,8 , Daisuke Aoki 1 , Kenshi Nakagawa 10 and
Takashi Shiina 11
1
2
3
4
5
6
7
8
9
10
11

*

Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo 1608582, Japan
Department of Pathology, Shiga University of Medical Science, Shiga 5202192, Japan
Department of Pathology, Keio University School of Medicine, Tokyo 1608582, Japan
Department of Surgery, Kidney Center, Tokyo Women’s Medical University, Tokyo 1628666, Japan
Department of Pediatric Surgery, Keio University School of Medicine, Tokyo 1608582, Japan
Department of Surgery, Keio University School of Medicine, Tokyo 1608582, Japan
Department of Anesthesiology, Saiseikai Kanagawaken Hospital, Kanagawa 2210821, Japan
Research Center for Animal Life Science, Shiga University of Medical Science, Shiga 5202192, JapanThe Corporation for Production and Research of Laboratory Primates, Ibaraki 3050003, Japan
Safety Research Center, Ina Research Inc., Nagano 3994501, Japan
Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine,
Tokai University School of Medicine, Kanagawa 2591193, Japan
Correspondence: iori71march@hotmail.co.jp or iori71march@a7.keio.jp; Tel.: +81-3-3353-1211;
Fax: +81-3-3353-0249

Received: 31 August 2019; Accepted: 30 September 2019; Published: 1 October 2019


Abstract: Uterus transplantation (UTx) is an option for women with uterine factor infertility to havea 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 andbiopsy of the transplanted uterus and immunoserological examinations were performed. All four
recipients survived for >3 months after surgery, but continuous menstruation did not resume, althoughtemporary 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: uterus transplantation; uterine transplantation; cynomolgus macaque; uterine factor
infertility; allogeneic uterus transplantation; rejection

J. Clin. Med. 2019, 8, 1572; doi:10.3390/jcm8101572

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1. Introduction
Uterus transplantation (UTx) has become a potential option for women with uterine factor
infertility to have a child. Data accumulated from animal studies for more than a decade have led toclinical application of UTx. Brännström et al. described the first human delivery after UTx with a living
donor in 2014 [1] and Ejzenberg et al. achieved the first live birth after UTx from a deceased donor in2017 [2]. These important achievements have attracted considerable attention worldwide, and many
teams have recently aimed for clinical application of UTx with accumulation of basic experimental
data. However, UTx is still in an experimental stage and there are many clinical and technical issues
to be resolved [3]. Therefore, careful accumulation of data in animal experiments is needed for fullestablishment of clinical application of UTx because the technology associated with UTx has notbeen standardized.
Animal studies on UTx have been performed in mice, rats, rabbits, dogs, pigs, sheep,and non-human primates [4]. Delivery after allogeneic UTx has only been achieved in rats [5]and sheep [6]. There are only a few reports on UTx in non-human primates, and delivery after
allogeneic UTx in these species has yet to be attained because of specific problems and limitations [7,8].
However, UTx studies in non-human primates are likely to provide important preclinical data for
resolution of problems in UTx in humans because these animals are anatomically and physiologicallysimilar to humans.
Our team in Japan has accumulated a large UTx research data archive using cynomolgus
macaques [9], which are commonly used as experimental models because of their anatomic andphysiologic similarity to humans. This allows data to be extrapolated to humans, except that both thesuperficial and deep uterine veins connect to the external iliac veins in cynomolgus macaques [10].
Also, the body size of female cynomolgus macaques used in our laboratory is similar to that of humanneonates and the weight is approximately 2.5–4 kg, which is a significant disadvantage compared withother non-human primate models, such as baboons [11]. Therefore, the challenge with this species
is the need for delicate surgical techniques for dissection and vascular anastomosis for their smaller
vessels. We previously succeeded in delivery after autologous UTx in cynomolgus macaque andestablished an autologous UTx animal model [12]. Thereafter, we tried to establish an allogeneic UTxmodel with living donors [13], but we encountered many cases of massive intraoperative bleeding anddeath due to hemorrhage intraoperatively and postoperatively.
With this background, we decided to move on to establishment of a surgical technique forallogeneic UTx, assuming recovery of a uterus from a deceased donor, to enable procurement ofwider and longer vessels; consequently, the surgical procedures and model preparation are morestable, in comparison with a living donor. As described in a previous report [14], we succeeded in
short-term survival after allogeneic UTx and establishment of a stable allogeneic UTx model withprocurement of common iliac vessels or aorta and inferior vena cava from a donor cynomolgus macaque.
However, this report had the important limitation that long-term outcomes, including immune system
suppression and postoperative organ rejection, were not evaluated. In preliminary allogeneic UTx withliving cynomolgus macaque donors [13], we were unable to overcome uterine rejection after surgery.
Therefore, as a next step for establishment of an allogeneic UTx model, long-term follow-up and use of
immunosuppressants are important. Herein, we report long-term outcomes in the four cynomolgus
macaques with short-term survival after allogeneic UTx described in our previous report [14].
2. Materials and Methods
2.1. Animals
Four female cynomolgus macaques (Macaca fascicularis, age 5–9 years; average body weight,
3.83 ± 1.12 kg (mean ± standard deviation) after allogeneic UTx [14] were used in this study. The four
recipients and their donors were selected for compatible ABO blood type and a high degree ofpolymorphism in the major histocompatibility complex (MHC) gene. Negative blood type crossJ. Clin. Med. 2019, 8, 1572

3 of 19match and donor-specific antibody (DSA) before surgery between donor and recipient were confirmed.
The study was performed in accordance with the recommendations in the Guide for the Care and Useof Laboratory Animals of the National Research Council and approved by the Animal Care and Use
Committee of the Research Center for Animal Life Science, Shiga University of Medical Science, Japan(permit number: 2013-4-2).
2.2. MHC Typing
Total RNA was isolated from peripheral blood cells using TRIzol reagent (Invitrogen/Life
Technologies/Thermo Fisher Scientific, Carlsbad, CA, USA). Complementary DNA was synthesizedusing ReverTra Ace (Toyobo, Osaka, Japan) after treatment of isolated RNA with DNase I(Invitrogen/Life Technologies/Thermo Fisher Scientific). Six primer pairs for MHC class I (Mafa-A
and Mafa-B), Mafa-DRB, Mafa-DQA1, Mafa-DQB1, Mafa-DPA1 and Mafa-DPB1 loci were used
separately for RT-PCR amplification [15,16]. In addition to these primer sets, we also designed 50
fusion primers that contained the 454 Titanium adaptor, key and multiple identifier sequences ineach locus. After RT-PCR amplification using high fidelity KOD FX polymerase (Toyobo), massively
parallel pyrosequencing of the PCR products was carried out using the GS Junior system andamplicon sequencing protocol (Roche, Basel, Switzerland). MHC genotypes were assigned bycomparing sequences to known MHC allele sequences in the Immuno Polymorphism Database(http://www.ebi.ac.uk/ipd/index.html) [17] using GS Reference Mapper ver. 3.0. The mapping
parameter was set to a perfect match between the read and reference sequences to avoid mismappingamong Mafa loci and contamination of in vitro generated PCR crossover amplicons [18]. If short-readsequences were not mapped to a reference sequence, a consensus sequence output from GS Reference
Mapper ver. 3.0 was used as a reference sequence for secondary mapping [19]. MHC genes of the four
recipients and donor pairs are shown in Table S1.
2.3. Uterus Transplantation
Operative anesthesia and surgical procedures in donors and recipients were performed aspreviously described [14]. In brief, animals were intubated and operated on under general anesthesia
with ventilation maintained by isoflurane inhalation (0.5%–1.5%; Abbot Japan, Tokyo, Japan).
All animals received 25 mg/kg of cefazolin every 3 h as antibiotic prophylaxis from initiation of
the operation. In donor surgery, thoracolaparotomy was performed, and the iliac vessels and thosesurrounding the uterus were dissected in cases 1 and 2. For further improvement of the surgicalprocedure based on the experience in cases 1 and 2, additional dissection including the abdominal
aorta/inferior vena cava (IVC) above the left renal vein and ovarian veins was performed in cases 3 and 4.
The ovaries of donors were removed in situ, but oviducts of donors were not removed. An anticoagulant(heparin, 1000 IU) was administered intravenously 5 min before cross-clamping of the abdominal aorta.
The uterine grafts were perfused with 200–350 mL of cold histidine-tryptophan-ketoglutarate solution(HTK® ; Custodiol® , Nordmedica, Gentofte, Denmark) via a perfusion catheter (22-G intravenous
needle; Terumo Corp., Tokyo, Japan) in the femoral or external iliac artery. Then, en bloc retrieval of
the uterus with the adnexa, ovarian vein, and proximal vagina was performed, including the iliac
vessels (cases 1 and 2) or infrarenal aorta/IVC (cases 3 and 4). The ovarian veins draining to the renalvein or IVC were left in the uterine graft side in cases 3 and 4. In recipient surgery, after entering theperitoneal cavity through a midline incision, a total simple hysterectomy was performed in advance,with preservation of the ovaries. Subsequently, iliac vessels on both sides (cases 1 and 2) or the
infrarenal abdominal aorta/IVC (cases 3 and 4) were exposed at a distance of approximately 2 cm.
The uterine graft was brought into the operative field and the vagina of the graft was anastomosedwith the vaginal vault of the recipient to fix the uterus in the pelvis. Before vascular anastomoses,
heparin (100 IU) was administered intravenously. In recipient surgery, end-to-side anastomosis withthe recipient’s and donor’s common iliac vessels (cases 1 and 2) or infrarenal aorta/ICV (cases 3 and 4)
was performed using continuous sutures (7–0 or 8–0 Prolene® , Johnson & Johnson; 9–0 Asflex® , Kono

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Seisakusho, Co., Ltd., Tokyo, Japan). After reperfusion, sutures of the retroperitoneum and roundligaments were performed before closing the abdominal incision. Normothermia was maintained
throughout the operation using heating lights and blankets in donors and recipients. Antibiotics and a
proton-pump inhibitor were administered after surgery.
2.4. Intraoperative Indocyanine Green Fluorescent Angiography
Intraoperative indocyanine green (ICG) (Diagnogreen 0.5%; Daiichi Pharmaceutical, Tokyo, Japan)
was injected intravenously to confirm the blood supply in the transplanted uterus and blood flowat the site of vascular anastomosis during surgery and in second-look surgery or autopsy [20–22].
Fluorescence intensity was displayed using a Photodynamic Eye Neo system (Hamamatsu Photonics
K.K., Hamamatsu, Japan).
2.5. Immunosuppressive Management
Severe rejections occurred in the first two transplants (cases 1 and 2) and the immunosuppressive
protocol was amended in cases 3 and 4. As induction treatment, animals received antithymocyte
globulin (ATG) (10 mg/kg; Thymoglobulin, Genzyme, Cambridge, MA, USA) intravenously on
postoperative day (POD) 0 (the day of surgery) in cases 1 and 2, and 20 mg/kg of ATG on POD 0
and POD 2 in cases 3 and 4. Maintenance treatment consisted of cyclosporine (CyA) (Sandimmune:
Novartis, Basel, Switzerland) given subcutaneously in cases 1 and 2, and tacrolimus (TAC) (Prograf;
Astellas Pharma, Tokyo, Japan) given intramuscularly in cases 3 and 4. These drugs were administeredfrom day 0 in case 1, day –2 in case 2, and day –1 in cases 3 and 4. The target trough levels for
CyA and TAC up to 1 month after surgery were planned to be in the ranges of 300–400 ng/mL and
15–20 ng/mL, respectively, and thereafter were adjusted as required. As another maintenance treatment,
mycophenolate mofetil (MMF) (40–100 mg/kg; Cellcept; Chugai Pharmaceutical, Tokyo, Japan) was
administered orally from recovery of appetite after surgery on POD 11 in case 1, POD 6 in case 3,
and POD 6 in case 4. MMF was discontinued on POD 22 in case 1 because the animal did not tolerate
the drug. For this reason, MMF was not given in case 2, but MMF was administered forcibly using an
orogastric catheter in cases 3 and 4, based on the severe rejections in cases 1 and 2. Methylprednisolone(10 mg/kg; Solu-Medrol; Pfizer, NY, USA) was injected intravenously on POD 0 and then injected
intramuscularly daily starting on POD 1. The dose of methylprednisolone was gradually tapered,and administration was completed on POD 14 in cases 1 and 2. In cases 3 and 4, methylprednisolone
was administered at daily doses of 0.5 and 0.2 mg/kg, respectively, until the end of observation.
If rejection occurred, steroid pulse therapy of 10 mg/kg methylprednisolone was administered andgradually tapered.
2.6. Flow Cytometry
After lysing red blood cells with BD Lysing buffer (BD Bioscience, Cat#:555899, Franklin
Lakes, NJ, USA), single cell suspensions of peripheral blood cells were immunostained withfluorescence-conjugated antibodies (Table S2). Dead cells were excluded using 7–aminoactinomycin
D (7–AAD; BD Pharmingen, Cat#: 559925, Franklin Lakes, NJ, USA). Isotype-matched antibodies
corresponding to each specific antibody were used as negative controls. Samples were analyzed using
a CytoFlex S instrument (Beckman Coulter Inc., Cat#: B75408, Brea, CA, USA).
2.7. Mixed Lymphocyte Reaction (MLR)
Heparinized peripheral blood was collected from macaques before and after transplantation.
After lysing red blood cells, 5 × 105 cells per well were cultured in an ELISPOT plate (Mabtech
AB, Cat#: FS-2122-2, Nacka Strand, Sweden) with 5 × 105 donor splenocytes. The culture wasusually performed in triplicate wells, but duplicate culture was used when the number of cells wasinsufficient. After culture for 3 days, the number of interleukin-2 (IL-2) positive spots was counted by
an ImmunoSpot analyzer (Cellular Technology, Shaker Heights, OH, USA). Stimulation indices (SI)J. Clin. Med. 2019, 8, 1572

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were calculated by the following formula: the sum of the number of spots in culture of blood cells anddonor splenocytes, divided by the number of spots in culture of blood cells only.
2.8. Donor-Specific Antibodies in Plasma of Macaques after Transplantation
After heat-inactivation, plasma collected from the transplanted macaques was diluted 10 times
with phosphate-buffered saline (PBS; Nacalai Tesque Inc., Cat#: 14249–25, Kyoto, Japan) before use.
The diluted plasma was added to a cell suspension of donor splenocytes as a primary antibody.
Fluorescein isothiocyanate (FITC)-conjugated goat polyclonal anti-mouse IgG (Nordic Immunological
Laboratories, Cat#: GAM/Fab/FITC, Copenhagen, Denmark) was used as a secondary antibody todetect the attached IgG on the surface of the donor cells. Phycoerythrin conjugated anti-CD20 antibody(Clone: 2H7, BioLegend, Cat#: 302306, San Diego, CA, USA), and allophycocyanin conjugated anti-CD3
antibody (Clone: SP34-2, BD Pharmingen, Cat#: 557597) were also used to study the cell type to which
the donor specific IgG attached. The cells were analyzed by FCM.
2.9. Postoperative Assessment
After the surgical procedure, animals were separately accommodated in a single cage andthe general condition (appetite, bowel movement, vomiting, urination, attitude) and laparotomy
wound site were evaluated daily. Laboratory assessments including hematology and blood chemistrywere performed three times per week for the first two postoperative weeks, two times per week
for two months, and weekly thereafter. To monitor for potential rejection after surgery, the sizeof the transplanted uterus and blood flow in the transplanted uterine artery were determined bytransabdominal ultrasonography under anesthesia. Transvaginal biopsy of the transplanted uterine
cervix and body and FACS, DSA and MLR analyses were routinely conducted monthly or when
considered necessary (whenever graft rejection was suspected). Animals were euthanized if severe
weakness, weight loss or abnormal behavior was seen or when determined to be necessary by veterinary
staff and investigators. Necropsy was performed immediately after euthanasia to evaluate the graft
and extrapelvic organs, including the brain, lung, heart, thymus, thymus, liver, gallbladder, spleen,pancreas, stomach, duodenum, jejunum, ileum, colon, rectum, kidney, bladder, bone marrow andlymph node.
2.10. Histological Evaluation, Immunohistochemistry and in Situ Hybridization
Formalin-fixed, paraffin-embedded liver biopsy tissues were sliced to a thickness of 3 µm andstained with hematoxylin and eosin (H&E). Elastica van Gieson (EVG) staining was also performed.
An automated staining system (Bond Max, Leica Biosystems) with a Bond Polymer Refine Detection kit(Leica Biosystems; catalog #: DS9800) was used for immunohistochemistry and in situ hybridization.
Immunohistochemistry was performed using an anti-CD20 monoclonal antibody (mouse; clone L26;
dilution, 1:200; Leica Biosystems; catalog #: CD20-L26-L-CE-H), an anti-CD3 monoclonal antibody(rabbit; clone SP7; dilution, 1:4; Abcam; catalog #: ab21703), and an anti-CD8 polyclonal antibody(rabbit; dilution, 1:400; Abcam; catalog #: ab4055). An EBER Probe (ready to use; Leica Biosystems;
catalog #: PB0589) was used for detection of Epstein–Barr virus-encoded mRNA by in situ hybridization.
Allograft rejection was evaluated histopathologically using a grading system for monitoring of rejection
proposed by a Swedish group [23,24], in which rejection of grades 1, 2 and 3 is described as mild,moderate and severe, respectively.
3. Results
3.1. Changes of Trough Levels of ImmunosuppressantsChanges of trough levels of CyA (cases 1 and 2) and TAC (cases 3 and 4) in maintenance treatment
are shown in Figure 1.

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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.

3.2. Overall Outcome
All four recipient animals survived for more than 3 months after surgery without any surgicalcomplications. The overall outcomes in these animals are summarized in Table 1.
Table 1. Summary of overall outcomes in recipients.

Case

1

DSA2

3

4

Survival
Days

196

126

140

104

Recovery of
Menstruation

TemporaryTemporary

None

None

Rejection
EpisodeSevereSevereMild–
Moderate

SevereDSA
Production

Positive

Positive

Negative

Positive

MLR Stimulation
Indexes(Pre-Transplant;
Maximum)

2.40; 40.0

19.9; 125.0

7.0; 33.7

5.9; 19.3

Cause of
Sacrifice

Major Pathological
Findings in Autopsy

Graft atrophy,
chronic
rejection

Atrophic uterus withhyalinized fibrosis and noendometrium
Grafted vessels with fibrous
occlusion and lymphocytes
infiltration

Graft atrophy,
chronic
rejection

Atrophic uterus withhyalinized fibrosis anduterine infection
Uterine stroma withlymphocytes infiltration andcapillary endotheliitis

Lower limbs
paralysis

Normal-sized uterus withmild lymphocytes
inflammation and a small
number of Civatte bodies in
the cervix
Nodular tumors diagnosed
as PTLD (diffuse large B-cell
lymphoma)

Swollen graft,chronic
rejection

Whole uterus with severe
endotheliitis, cervix withepithelial desquamation, andcorpus with obstructive
vasculitis and focal necrosis
Feeding vessels in grafted
aorta adventitia with severe
endotheliitis and obstruction

DSA; Donor-specific antibody; MLR; Mixed lymphocyte reaction.

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Oral administration of MMF (50 mg/kg, twice a day) was started on POD 11 because of recovered
appetite, but discontinued on POD 22 due to a decrease in appetite. On POD 32, an increased
inflammatory response (WBC 181 × 102 /µL, CRP 11.8 mg/dL) and elevated LDH (1076 IU/L) were
found. Swelling of the uterus was found despite blood flow in the uterine artery identified bytransabdominal ultrasonography, and rejection was suspected. However, rejection could not be
confirmed because samples that were appropriate for evaluation were not obtained from cervicalbiopsy specimens. The increased inflammatory response and LDH were improved by antibiotics;
however, a biopsy specimen from the uterine cervix on POD 70 showed stromal lymphocyte infiltration
with capillary endotheliitis, which was consistent with severe rejection (Figure 2A). Consequently,
steroid pulse therapy was used.

Figure 2. Macroscopic and histopathological findings in case 1. (A) Histopathological findings of abiopsy specimen from the uterine cervix on postoperative day (POD) 70. Severe stromal inflammationand capillary endotheliitis including lymphocytes and eosinophils were observed. Lymphocytes also
infiltrated into squamous epithelium and resulted in liquefaction degeneration (white arrow). Necroticchange 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 thetransplanted 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 withhemosiderin 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), whichsuggests mild rejection. Bar = 100 µm.

A second-look surgery was performed on POD 133 to assess the therapeutic effect. Subsequent
ICG fluorescence angiography showed no uterine blood flow, and a uterine biopsy specimen showedcoagulative necrosis, fibrosis with hyalinization, and lymphocyte infiltration with endotheliitis
in the myometrial tissue. Immunohistochemistry showed that CD8+ T-cells had infiltrated into
capillary vessels. (Figure 2B). We interpreted these findings as severe rejection with resistance toimmunosuppressive treatment. Administration of CyA was discontinued on POD 143. Systemic
conditions were good, but the uterus gradually shrank and was dissected on POD 196. Intraperitoneal

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findings showed a white shrunken uterus adhered to a wide range of the omentum and rectum,with left hydrosalpinx (Figure 2C). In vascular anastomotic sites, the grafted common iliac artery
had collapsed and no beat was detected. ICG fluorescent angiography showed no blood flow in thewhole uterus and in anastomosed grafted vessels (Figure 2D). Histologically, the uterus was severely
atrophic and had no endometrium. The myometrial area was replaced by hyalinized fibrosis in whichsmall vessels were occlusive. The main grafted vessels showed complete fibrous occlusion, CD8-richlymphocyte infiltration, and hemosiderin deposition (Figure 2E). The donor’s grafted oviduct showeddilation and CD8-rich lymphocyte infiltration, which also suggested rejection (Figure 2F).
3.4. Case 2
Administration of MMF was not planned in case 2 because of the postoperative decrease in appetite
in case 1. Blood flow in the uterine artery was identified by transabdominal ultrasonography on POD
20, but slight swelling of the uterus was found. However, menstruation resumed and follow-up was
performed without treatment. Biochemistry on POD 32 showed an increased inflammatory response(WBC 195 × 102 uL, CRP 14.6 mg/dL) and elevated LDH (918 IU/L). Rejection was suspected based on
the results of case 1. Consequently, second-look surgery was performed on POD 35. The uterus wasgrossly adhered to the omentum and was markedly swollen (Figure 3A). High adhesion was foundaround the adnexa. Histologically, myometrial tissue showed focal necrotic change. Endotheliitis with
CD8-rich lymphocyte infiltration and endothelial desquamation were observed in the myometrial
interstitium (Figure 3B). These findings suggested severe rejection. Consequently, steroid pulse therapy
and administration of ATG (20 mg/kg) were performed.

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) Histopathologicalfindings 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 ultrasonographyof 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 fluorescenceimaging 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.

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Pathological rejection including myometrial necrosis and endotheliopathy was still found in auterine biopsy on POD 70, indicating no therapeutic effect. Based on the above, improvement ofrejection was determined to be difficult, and administration of CyA was discontinued on POD 75. No
uterine blood flow was found on transabdominal ultrasonography on POD 97 and abscesses were
present in the uterine cavity (Figure 3C). Transvaginal drainage was performed and Escherichia coli was
identified in a bacterial culture test. A slightly elevated inflammatory response (WBC 111 × 102 uL,
CRP 1.7 mg/dL) was found in blood tests, but systemic conditions were good. After that, the uterus
gradually shrank and was dissected on POD 126. Similarly to case 1, the intraperitoneal findings werea white shrinking uterus adhered to a wide range of the greater omentum and rectum, with bilateralhydrosalpinx (Figure 3D). Vascular anastomotic sites could not be identified due to high adhesion.
The whole uterus was not imaged by ICG fluorescence angiography (Figure 3E). The removed uterus
showed atrophy with hyaline fibrosis. In the fibrotic area, CD8-rich lymphocyte infiltration wasobserved. The uterine cavity contained a lot of neutrophils, which was suggestive of uterine infection(Figure 3F). As for case 1, the vessels around grafted tubes showed CD8-rich endotheliitis.
3.5. Case 3
MMF was administered twice a day at 50 mg/kg using an orogastric catheter from POD 6. Bloodflow in the uterine artery was normal in ultrasound on POD 20 and no rejection was found in biopsy.
However, severe anemia occurred with Hb down to 4.4 g/dL from POD 38–61, as an adverse effect of
MMF. Consequently, MMF was withdrawn and transfusion was performed to treat anemia. Duringthat period, trough concentrations of tacrolimus were low (Figure 1). Biochemistry on POD 61 showed
an increased inflammatory response (WBC 169 × 102 uL, CRP 20.8 mg/dL) and elevated LDH (515 IU/L),
and rejection was suspected based on the results in cases 1 and 2. Consequently, the dose of tacrolimus
was increased from POD 62, MMF was readministered at 30 mg/kg twice a day, and steroid pulse
therapy was started. The inflammatory response and LDH improved, but the trough concentrations oftacrolimus were poorly controlled and high until POD 116 (Figure 1). Uterine blood flow was poor in
ultrasonography on POD 83.
A uterine biopsy on POD 83 showed stromal CD8-rich lymphocyte infiltration and liquefaction
degeneration with a small number of Civatte bodies (Figure 4A,B). The biopsy also included myometrial
tissue, which was atrophic and had a small area of hyalinization. These findings indicated at least
moderate rejection, and this was improved to mild rejection based on a biopsy on POD 118. However,
the macaque developed right eyelid swelling on POD 120 (Figure 4C) and bilateral leg paralysis
on POD 129, resulting in buttock decubitus due to leg paralysis. Consequently, the macaque was
euthanized and dissected on POD 140. Laparotomy and thoracotomy results showed a red-coloreduterus of normal size and adhesion around the left adnexa (Figure 4D). Tumors of 4 cm and 7 cm
were found near the abdominal aorta and the anterior mediastinum, respectively, and tumors werealso found in the left adrenal gland and right eyelid. Histologically, the uterine cervix showed mild
lymphocyte infiltration, liquefaction degeneration, and a small number of Civatte bodies, which still
indicated mild rejection. However, all nodular tumors were composed of large-sized atypical B-cells
and focal necrotic changes. These tumor cells were positive for CD20 and EBER-ISH, but negative forCD3 and CD8. The diagnosis was monomorphic post-transplant lymphoproliferative disorder (PTLD),
diffuse large B-cell lymphoma (Figure 4E,F). Possible causes of bilateral leg paralysis were spinal cordischemia and stenosis induced by vascular retraction by PTLD and direct invasion of the spinal cord.
Serum EB virus IgG antibody was positive before UTx.

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Figure 4. Macroscopic and histopathological findings in case 3. (A,B) Histopathological findings ofa biopsy specimen from the uterine cervix on POD 83. Lymphocytes infiltrated the epithelium andstroma 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) Histopathologicalfindings of a resected nodular tumor, showing monomorphic large-sized lymphoid cells with abundantnucleoli. H&E stain, 200x. (E). Bar = 100 µm. These atypical cells were positive for CD20 and EBER-ISH(window). (F). Bar = 200 µm.

3.6. Case 4
Based on the PTLD in case 3, the dose of MMF on POD 6 was decreased from 40 to 20 mg/kg(twice a day, using an orogastric catheter) and the maintenance dose of methylprednisolone wasdecreased from 0.4 to 0.2 mg/kg in case 4. When starting administration of ATG, ganciclovir (20 mg/kg;
Denosine; Mitsubishi Tanabe Pharma, Osaka, Japan) was also injected intravenously. Transabdominal
ultrasonography on POD 13 showed a uterus of normal size and good blood flow in the uterine artery.
Histologically, liquefaction degeneration was mild; however, perivascular lymphocyte infiltrationand endotheliitis were clearly evident. Based on the endotheliitis, this condition was diagnosed asmoderate rejection. No increased inflammatory response or LDH was found in biochemistry tests.
Consequently, steroid pulse therapy was used. Transabdominal ultrasonography on POD 25 showedslight uterine swelling. A biopsy specimen showed similar rejection features to those on POD 13,
with perivascular fibrosis and edematous changes. Steroid pulse therapy was administered again,
ATG was given at 20 mg/kg, and MMF was increased to 30 mg/kg twice a day. In a biopsy on POD
41, obstructive venulitis and endotheliitis of small to middle size arteries were seen, in addition toworsening of capillary endotheliitis, and perivascular fibrosis (Figure 5A). Biochemistry findings on
POD 65 showed an elevated inflammatory response (WBC 206 × 102 uL, CRP 17.1 mg/dL). This was
improved by antibiotics, leading to a good systemic condition.

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Figure 5. Macroscopic and histopathological findings in case 4. (A) Histopathological findings of auterine 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 theabdomen in autopsy on POD 104, showing a whitish swollen transplanted uterus. Severe adhesion at asite 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) showedthat 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 theanastomotic 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 findingsof 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.

The uterus subsequently became more swollen and was dissected on POD 104, after it was decided
that rejection could not be relieved. Laparotomy results showed a markedly swollen white uterus thatadhered to the omentum and to vascular anastomotic sites surrounding the aorta and IVC (Figure 5B).
The whole uterus was not imaged, but blood flow at vascular anastomotic sites distal from the aorta
was not detected by ICG fluorescence angiography. A hypertrophic myometrium was found in theresected uterus, and no endometrium was detected. An abscess was found in the uterine cavity
and Escherichia coli was identified in a bacterial culture test. In the resected aorta, the donor-grafted
intravascular space in the vascular anastomotic sites was almost stenosed (Figure 5C) and thrombi were
found in the grafted intravascular space from the aorta to bilateral common iliac arteries (Figure 5D).
Severe endotheliitis was observed in the whole uterus, along with epithelial desquamation in the cervix(Figure 5E), obstructive vasculitis and focal necrosis in the corpus. The donor’s oviducts had severe
lymphocyte infiltration and epithelial apoptotic changes. In the grafted aorta, lymphocytes were notattached to the endothelium of the aorta directly; however, its feeding vessels in the vascular adventitia
showed severe endotheliitis, mainly due to CD8+ T-cells, and some showed obstruction (Figure 5F).

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4. Recovery of Cyclicity
Temporary menstruation resumed from POD 45 and POD 96 in case 1 and POD 17 in case 2,
but amenorrhea then continued in both animals. Menstruation did not resume after surgery in cases 3
and 4.
4.1. Changes in Peripheral Lymphocyte Counts
Induction treatment and additional ATG administration decreased lymphocyte counts immediately
after surgery (Figure 6). Due to differences in ATG doses, this decrease in cases 3 and 4 was prolonged
compared to that in cases 1 and 2. Changes of peripheral CD3+ , CD20+ and NK cell (CD3– CD8+ CD16+ )counts and the CD4+ /CD8+ cell ratio are shown in Figure 7. Immediately after induction treatment,
counts of CD3, CD20 and NK cells decreased, but then gradually increased in correspondence torejection. The CD4+ /CD8+ ratio showed a tendency to decrease. Regulatory T cells (CD4+ FOXP3+ )had no relationship with rejection (data not shown).

Figure 6. Changes in peripheral lymphocyte counts. Lymphocyte counts decreased after administrationof antithymocyte globulin (ATG). The nadir lymphocyte counts in cases 3 and 4 were prolonged bydifferences in ATG doses, in comparison with counts in cases 1 and 2.

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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.

4.2. Leukocyte Proliferation Against Donor or Third-Party Antigens
The MLR stimulation index against donor and third-party antigens was determined after surgery(Figure 8). All animals exhibited elevation of this index.

Figure 8. Mixed lymphocyte reaction (MLR) stimulation indexes pre- and post-transplantation.
(A) Case 1. (B) Case 2. (C) Case 3. (D) Case 4.

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14 of 194.3. Antidonor Antibody Production
All animals were seronegative for DSA before transplantation. DSAs against MHC class I and II
were detected in conjunction with uterine rejection on PODs 46 (data not shown), 35 and 13 in cases 1,
2 and 4, respectively, after which the DSA titer increased (Figure 9). In case 3, DSAs against MHC class
I and II were negative.

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.

5. Discussion
UTx is now conducted in humans in several countries, but is still considered to be in theexperimental stage since there are remaining clinical and technical issues to be resolved [25]. Therefore,
development of allogeneic animal models of UTx is required for improvement of clinical UTx, including
establishment of suitable immunosuppressants, diagnosis of rejection, monitoring of a grafted uterus,
uterine antigenicity, and teratogenicity induced by immunosuppressants. Experiments in non-humanprimates are particularly appropriate for extrapolation to clinical application of UTx because theseanimals are anatomically and physiologically similar to humans. However, there are some problems
and limitations with experimental use of non-human primates for UTx [7,8]. Female macaques are
small compared to humans, and complex vascular dissection of the pelvic floor is required in UTx.
Therefore, fine surgical techniques are required in a macaque model. Postoperative management
also has difficulties. Control of the blood level of immunosuppressive agents is important because ofeffects on graft survival, but it is difficult to administer these agents orally in non-human primates asscheduled, and delivery via enteral feeding is problematic because of marked anorexia after invasive
surgery and side effects of the agents. Intravenous administration is simple in humans, but difficult toJ. Clin. Med. 2019, 8, 1572

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control in animal models. These difficulties also cause marked postoperative weight loss that may
not be recovered by supplementation with oral nutrients alone, and too much weight loss leads to an
endpoint of the study in most settings. Moreover, postoperative examinations including echography
and biopsy cannot be easily performed because sedation is required, and blood test items are also
limited in non-human primates, compared to humans.
In this study, long-term outcomes after allograft were examined in four cynomolgus macaques
used in our previous study to establish the allograft procedure. All four macaques developed rejection
and three underwent hysterectomy because uterine function could not be recovered due to irreversible
chronic rejection. There are case reports of human UTx with rejection [23,26–28] but no case of
hysterectomy due to refractory rejection. Therefore, clinicopathological features of rejection in themacaques in follow-up are important findings for solving problems from the perspective of basic
medicine in UTx. The clinical features of severe rejection found in this study are summarized in thefollowing paragraphs.
First, an increased WBC and CRP inflammatory response due to rejection and elevated LDH
due to cell collapse were found. In rejection or elimination of conventional life-supporting organtransplants, clinical symptoms and biochemical data change immediately. In contrast, the uterus is nota life-supporting organ and rejection is not immediately life-threatening and frequently asymptomatic;
therefore, there are no clear findings showing rejection. The biochemical findings above may be one
indicator; however, these findings are often caused by irreversible rejection and it may be difficult torecover uterine function in such a case.
Second, the uterus is swollen by rejection and then shrinks over time. In the course, inflammatory
edema occurs due to rejection, followed by cell injury and necrotic cell death, and granulation tissues
and fibrils are formed, resulting in atrophy. In cases 1 and 2, these histopathologic findings were
found, but in case 4, the uterus swelled but did not shrink. However, endotheliopathy and vascularocclusion were observed, and the uterus was assumed to have atrophied due to ischemia over time.
This assumption is suggested by our previous results showing that the uterus of cynomolgus macaqueshrinks after a warm ischemia time of 8 h [29,30].
Third, continuous rejection sometimes promotes intrauterine infection. The uterine cavity is incontact with the outside of the body through the vagina; therefore, intrauterine infection may occur.
Since organ transplant recipients are treated with immunosuppressant drugs, they have higher risks for
infection than general patients. Furthermore, rejection and ischemia disrupt phylaxis functions in theuterus and enhance the risk for infection. In cases 2 and 4, intrauterine infection occurred, and E. coli
was detected in both. Hygiene control of reared cynomolgus macaques is limited and E. coli fromfeces probably contaminated the uterus. Also, a UTx patient from Sweden had repeated intrauterine
infection and the uterus had to be resected [31].
Fourth, rejection causes thrombus formation. An intravascular thrombus is often caused bysurgical procedures of vascular anastomosis and sometimes by blood flow stasis because of occlusion ofsmall and middle-size vessels in the rejected organ due to rejection-induced endotheliopathy. In case 4,
thrombi were found from the vascular anastomotic site of the grafted aorta to the iliac artery and severe
endotheliopathy occurred. Furthermore, inflammatory cells were detected in the outer membranes of
the grafted aorta, which suggests that rejection in vessels is probably the cause of thrombi.
Fifth, interestingly, rejection occurred in the oviducts, as well as in the uterus. In cases 1 and 2,
lymphocytic infiltration was found in the oviducts of donors. In human UTx, the donor’s oviducts areoften removed in transplantation, despite retention of the oviduct in donors having an advantage of
maintaining the option of natural pregnancy. However, hydrosalpinx was evoked in cases 1 and 2 dueto the remaining oviduct, and this may cause infertility; therefore, surgery with oviduct resection iscurrently preferable.
Finally, the histopathological findings in rejection due to UTx in cynomolgus macaques included
frequent onset of endotheliopathy, in addition to those based on clinical criteria for uterine rejection bya Swedish group [23,24]. Simultaneously, DSAs against MHC class I and II are likely to be positive.

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Therefore, additional induction treatment with rituximab and a triple regimen may be necessary to
inhibit B cells from humoral rejection. Histopathology showed endotheliopathy in the interstitium
more than the epithelium; therefore, humoral rejection may be overlooked if interstitial biopsy is
not performed.
Postoperative immunosuppression for prevention of rejection due to organ-specific antigenicity isimportant in organ transplantation. Antigenicity of the uterus is unclear, but in human UTx many
patients have received immunosuppressant protocols similar to those used in renal transplantation.
There have been no reports of serious rejection of the uterus; therefore, the uterus is considered to
be an organ that is unlikely to develop rejection. However, from our previous results [13] and theoutcomes of this study, we conclude that the uterus of cynomolgus macaques is likely to be rejected.
Furthermore, in transplantation in cynomolgus macaques, higher doses of immunosuppressants thanthose used in humans are required, and it is necessary to increase the blood trough concentrationsof calcineurin inhibitors to higher than those in humans [32–35] or inhibiting T cells with a higher
ATG dose [36]. Also, enhanced immunosuppression may cause PTLD, as in case 3. Therefore, strict
postoperative control of immunosuppressants with limited drug administration and examination incynomolgus macaques is difficult.
Our results show that studies in non-human primate models have specific problems and limitations,and recognition is required that that results may not always correspond directly to those in humans.
However, development of an allogeneic UTx model in a non-human primate is still important foraddressing clinical issues in human UTx, and further validation and outcomes of UTx in such models
will contribute to successful clinical use of UTx, in which current results indicate exciting developments.
The outcomes of these procedures are likely to be improved by accumulation of data from further
animal studies, including in non-human primates.
In conclusion, allograft of the uterus of cynomolgus macaques resulted in clinicopathologicalfindings of increased inflammatory response, swollen and subsequently shrunken uterus, intrauterine
infection, intravascular thrombus formation, oviductal rejection, and a DSA-positive status.
Postoperative management and monitoring of macaques are limited, but the uterus of cynomolgus
macaques was found to be likely to develop rejection and to have high antigenicity. Therefore,
adjustment of protocols for immunosuppressive drugs may be necessary to produce a stable uterine
allograft model in cynomolgus macaques. However, the current results are important for resolution ofremaining clinical questions in human UTx by producing a scientifically verifiable model of UTx in a
non-human primate.
Supplementary Materials: The following are available online at http://www.mdpi.com/2077-0383/8/10/1572/s1,
Table S1. MHC genes of four recipient and donor pairs, Table S2. Antibodies for flow cytometry.
Author Contributions: Conceptualization, I.K. (Iori Kisu); methodology, I.K. (Iori Kisu), Y.K., Y.Y., K.N. and
T.S. (Takashi Shiina); validation, I.K. (Iori Kisu), H.I., K.E., Y.K. Y.Y., and Y.M. (Yusuke Matoba); formal analysis,
I.K. (Iori Kisu), H.I., K.E. T.S. (Takako Sasamura), H.U., K.N. and T.S. (Takashi Shiina); investigation, I.K. (Iori
Kisu), Y.K., Y.Y., K.M., H.O., Y.M. (Yusuke Matoba), M.A., Y.S., I.I., I.K. (Ikuo Kawamoto), C.I., T.N. and H.T.; data
curation, I.K. (Iori Kisu), H.I. and K.E.; experimental support in animals, I.I., I.K. (Ikuo Kawamoto), C.I., T.N. and
H.T.; writing—original draft preparation, I.K. (Iori Kisu); writing—reviewing & editing, H.I. and K.E.; supervision,
K.O., M.E., K.B., D.A., K.N. and T.S. (Takashi Shiina); funding acquisition, I.K. (Iori Kisu), K.O., K.N. and T.S.
(Takashi Shiina). All authors read and approved the final manuscript.
Funding: This work was supported by the Adaptable and Seamless Technology Transfer Program throughTarget-Driven Research and Development (A-STEP), AMED (AS2525033N), and the Japan Society for the
Promotion of Science (JSPS) KAKENHI (Grant Number 26713050).
Acknowledgments: This work was supported by the Adaptable and Seamless Technology Transfer Program
through Target-Driven Research and Development (A-STEP), AMED (AS2525033N), and the Japan Society for the
Promotion of Science (JSPS) KAKENHI (Grant Number 26713050).
Conflicts of Interest: The authors declare no conflict of interest.
Details of Ethics Approval: This study was approved by the Animal Care and Use Committee of the ResearchCenter for Animal Life Science, Shiga University of Medical Science, Japan (permit number: 2013-4-2).

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