Abstract
For pituitary regenerative medicine, the creation of a hypophyseal model in monkeys is necessary to conduct future preclinical studies; however, previous studies reported that hypophysectomy ...in monkeys is not always safe or satisfactory. This study aimed to create a hypophyseal dysfunction model in a cynomolgus monkey using a safer surgical technique and establish the protocol of pituitary hormone replacement therapy for this model. Surgical resection of the pituitary gland of a 7.8-year-old healthy adult cynomolgus male monkey weighing 5.45 kg was performed to create a hypophyseal dysfunction model for future regenerative studies. Endoscopic transoral transsphenoidal surgery was used to perform hypophysectomy under navigation support. These procedures were useful for confirming total removal of the pituitary gland without additional bone removal and preventing complications such as cerebrospinal fluid leakage. Total removal was confirmed by pathological examination and computed tomography. Hypopituitarism was verified with endocrinological examinations including stimulation tests. Postoperatively, the monkey’s general condition of hypopituitarism was treated with hormone replacement therapy, resulting in long-term survival. The success of a minimally invasive and safe surgical method and long-term survival indicate the creation of a hypophyseal dysfunction model in a cynomolgus monkey; hence, this protocol can be employed in the future.
Uterus transplantation (UTx) is a potential option for women with uterine factor infertility to have a child. The clinical features indicating irreversible rejection of the uterus are unknown. In our ...experimental series of allogeneic UTx in cynomolgus macaques, six female macaques were retrospectively examined, which were unresponsive to treatment with immunosuppressants (i.e. irreversible rejection). Clinical features including general condition, hematology, uterine size, indocyanine green (ICG) fluorescence imaging by laparotomy, and histopathological findings of the removed uterus were evaluated. In all cases, general condition was good at the time of diagnosis of irreversible rejection and thereafter. Laboratory evaluation showed temporary increases in white blood cells, lactate dehydrogenase and C-reactive protein, then these levels tended to decrease gradually. In transabdominal ultrasonography, the uterus showed time-dependent shrinkage after transient swelling at the time of diagnosis of irreversible rejection. In laparotomy, a whitish transplanted uterus was observed and enhancement of the transplanted uterus was absent in ICG fluorescence imaging. Histopathological findings in each removed uterus showed hyalinized fibrosis, endometrial deficit, lymphocytic infiltration and vasculitis. These findings suggest that uterine transplantation rejection is not fatal, in contrast to rejection of life-supporting organs. Since the transplanted uterus with irreversible rejection atrophies naturally, hysterectomy may be unnecessary.
There are few effective antimicrobial agents against Balantidium coli infection. The effect of paromomycin sulfate (PS) against B. coli was confirmed in this study of 596 captive cynomolgus monkeys. ...In several trials, the minimum dose and duration of oral administration of PS were 25 mg/day for 5 + 5 days, with a 2-day withdrawal interval. To facilitate daily PS administration, pumpkin cakes supplemented with PS were made, which not only resulted in precise effects but also increased the efficiency of preparation and administration of PS by the animal care staff. No cysts or trophozoites were detected at 14 or 16 days after the last treatments. There were no obvious differences in blood and biochemical parameters between before and after administration of PS. These results indicate that PS is effective for elimination of B. coli without hematological side effects. These data could contribute to the control of microbiological pathogens during veterinary care and colony management in primate facilities.
Abstract
STUDY QUESTION
How long is the allowable warm ischemic time of the uterus and what morphological and biochemical changes are caused by uterine ischemia/reperfusion injury in cynomolgus ...macaques?
SUMMARY ANSWER
Warm ischemia in the uterus of cynomolgus macaques is tolerated for up to 4 h and reperfusion after uterine ischemia caused no further morphological and biochemical changes.
WHAT IS KNOWN ALREADY
Uterus transplantation is a potential option for women with uterine factor infertility. The allowable warm ischemic time and ischemia/reperfusion injury of the uterus in humans and non-human primates is unknown.
STUDY DESIGN, SIZE, DURATION
This experimental study included 18 female cynomolgus macaques with periodic menstruation.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Animals were divided into six groups of three monkeys each: a control group and groups with uterine ischemia for 0.5, 1, 2, 4 and 8 h. Biopsies of uterine tissues were performed before blood flow blockage, after each blockage time, and after reperfusion for 3 h. Blood sampling was performed after each blockage time, and after reperfusion for 5, 15 and 30 min for measurement of biochemical data. Resumption of menstruation was monitored after the surgical procedure. Morphological, physiological and biochemical changes after ischemia and reperfusion were evaluated.
MAIN RESULTS AND THE ROLE OF CHANCE
Mild muscle degeneration and zonal degeneration were observed in all animals subjected to warm ischemia for 4 or 8 h, but there were no marked differences in the appearance of specimens immediately after ischemia and after reperfusion for 3 h in animals subjected to 4 or 8 h of warm ischemia. There were no significant changes in any biochemical parameters at any time point in each group. Periodical menstruation resumed in all animals with warm ischemia up to 4 h, but did not recover in animals with warm ischemia for 8 h with atrophic uteri.
LIMITATIONS, REASON FOR CAUTION
Warm ischemia in actual transplantation was not exactly mimicked in this study because uteri were not perfused, cooled, transplanted or reanastomosed with vessels. Results in non-human primates cannot always be extrapolated to humans.
WIDER IMPLICATIONS OF THE FINDINGS
The findings suggest that the tolerable warm ischemia time in the uterus is expected to be longer than that in other vital organs.
STUDY FUNDING/COMPETING INTEREST(S)
This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 26713050. None of the authors has a conflict of interest to declare.
Familial hypercholesterolemia (FH) is an inherited autosomal dominant disorder that is associated with a high plasma level of low-density lipoprotein (LDL) cholesterol, leading to an increased risk ...of cardiovascular diseases. To develop basic and translational research on FH, we here generated an FH model in a non-human primate (cynomolgus monkeys) by deleting the LDL receptor (LDLR) gene using the genome editing technique. Six LDLR knockout (KO) monkeys were produced, all of which were confirmed to have mutations in the LDLR gene by sequence analysis. The levels of plasma cholesterol and triglyceride were quite high in the monkeys, and were similar to those in FH patients with homozygous mutations in the LDLR gene. In addition, periocular xanthoma was observed only 1 year after birth. Lipoprotein profile analysis showed that the plasma very low-density lipoprotein and LDL were elevated, while the plasma high density lipoprotein was decreased in LDLR KO monkeys. The LDLR KO monkeys were also strongly resistant to medications for hypercholesterolemia. Taken together, we successfully generated a non-human primate model of hypercholesterolemia in which the phenotype is similar to that of homozygous FH patients.
Introduction
The objective of this study was to examine the allowable warm ischemic time and pathological changes due to ischemia and reperfusion injury in the uterus of the cynomolgus monkey as a ...model for uterus transplantation.
Material and methods
Six female cynomolgus monkeys were used in the study. The uterus was resected from the vaginal canal and connected through the bilateral ovarian and uterine arteries and veins only. One animal was used as a control. In the other five animals, the bilateral uterine and ovarian vessels were clamped for 0.5, 1, 2, 4 and 8 h, respectively. Biopsy of the smooth muscle tissue of corpus uteri was performed after each ischemic time and after subsequent reperfusion for 3 h. Biopsy samples were observed by light and electron microscopy. Menstruation recovery was monitored.
Results
There were no particular findings in both light and electron microscopy after ischemia for up to 2 h and after subsequent reperfusion. There were no marked changes after ischemia for 4 h, but dilated nuclear pores and rough endoplasmic reticulum swelling were found after reperfusion. These changes also occurred, along with mitochondrial swelling and cristae loss after ischemia for 8 h, and plasma membrane loss, nuclear fragmentation and chromatin condensation were found after reperfusion. Periodical menstruation restarted in all animals with ischemia up to 4 h, but the animal with ischemia for 8 h had amenorrhea and uterine atrophy.
Conclusions
The uterus of the cynomolgus monkey tolerates warm ischemia for up to 4 h.
No study has reported an animal model of uterus transplantation (UTx) using cynomolgus macaques. We aimed to establish a surgical technique of allogeneic UTx assuming the recovery of a uterus from a ...deceased donor in cynomolgus macaques. Four allogeneic UTxs were performed in female cynomolgus macaques. Donor surgeries comprised en bloc recovery of organs with iliac vessels on both sides, and/or abdominal aorta/vena cava after sufficient perfusion from one femoral artery or external iliac artery. Before perfusion, 150 mL of whole blood was obtained from the donor for subsequent blood transfusion to the recipient. Four uterine grafts were orthotopically transplanted to recipients. End-to-side anastomosis was performed to the iliac vessels on one side in case 1 and iliac vessels on both sides in case 2; aorto-aorto/cavo-caval anastomosis was performed in cases 3 and 4. Arterial blood flow of the uterine grafts was determined by intraoperative indocyanine green (ICG) angiography. ICG angiography results showed sufficient blood flow to all uterine grafts, and anaemia did not progress. Under appropriate immune suppression, all recipients survived for more than 90 days post-transplantation, without any surgical complications. We describe a surgical technique for allogeneic UTx in cynomolgus macaques.
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.
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.
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