Experimental techniques for the development of a uterus transplantation model in cynomolgus macaques.

Uterus transplantation (UTx) is now a treatment for women with uterine factor infertility to have a child. However, UTx is still largely at the experimental stage, and many medical issues remain unsolved. Therefore, adequate studies in large animals including non-human primates are required for validation of these issues. UTx research, especially in non-human primates, can provide important information for its full establishment in humans due to the anatomical and physiological similarities between the two. We accumulated data from UTx studies using cynomolgus macaques since 2009 and established autologous and allogeneic UTx models which led to deliveries after performing the procedure. In this paper, we summarized key points to develop UTx models in cynomolgus macaques based on our experience. UTx models in non-human primates can surely contribute new and beneficial knowledge in this field and can be useful for the further development of UTx in humans.

Duration of Vascular Endothelial Growth Factor Suppression After Intravitreal Injection of Brolucizumab and Aflibercept in Macaque Eyes.

Purpose: To compare the duration of vascular endothelial growth factor (VEGF) suppression in the aqueous humor of macaque eyes after intravitreal injection of brolucizumab and aflibercept. Methods: Clinical dose of intravitreal brolucizumab (IVBr; 6.0 mg/50 µL) or intravitreal aflibercept (IVA; 2 mg/50 µL) was injected into the right eye of each of 8 macaques. Aqueous humor samples (150 µL) from both eyes were obtained just before injection and on days 1, 3, 7, 14, 21, 28, 42, 56, 84, and 112 after IVBr injection or IVA injection. VEGF concentrations were measured using enzyme-linked immunosorbent assays. Results: In the injected eyes, the mean VEGF suppression durations (range) were 4.9 (3-8) weeks for IVBr injection and 6.8 (6-8) weeks for IVA injection (P = 0.04). The VEGF concentrations returned to the preinjection level in the aqueous humor at 12 weeks both after IVBr and IVA injection. In the noninjected fellow eyes, the aqueous VEGF concentrations had decreased least at 1 day after IVBr injection and at 3 days after IVA injection, but were still detectable. The VEGF concentrations in the fellow eyes returned to the preinjection level in the aqueous humor at 1 week after IVBr injection and at 2 weeks after IVA injection. Conclusions: The duration of VEGF suppression in the aqueous humor after IVBr injection may be shorter than that after IVA injection, which may be related with clinical usage.

Generation of a familial hypercholesterolemia model in non-human primate.

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.

Pharmacokinetics of Intravitreal Vancomycin and Ceftazidime in Silicone Oil-Filled Macaque Eyes.

Purpose: This study evaluated the pharmacokinetics of intravitreal vancomycin and ceftazidime in the aqueous humor of macaque eyes filled with silicone oil in the vitreous cavity. Methods: Intravitreal vancomycin (1 mg/0.1 mL) and ceftazidime (2 mg/0.1 mL) were injected into four normal macaque eyes, four vitrectomized aphakic macaque eyes, and four previously vitrectomized aphakic macaque eyes filled with silicone oil (silicone oil-filled eyes). Aqueous humor samples (0.1 mL) were obtained just before injection and at 2 and 5 hours and 1, 2, 3, 5, 7, and 10 days after injection. In each group, corneal endothelial cell density (ECD) measurements and electroretinogram (ERG) recordings were obtained before injection and after 1 month. Results: The half-lives of vancomycin in the aqueous humor of normal, vitrectomized, and silicone oil-filled eyes were 29.4, 21.1, and 6.8 hours, respectively, and those of ceftazidime were 20.4, 5.2, and 3.1 hours, respectively. The maximum vancomycin aqueous humor concentrations of normal, vitrectomized, and silicone oil-filled eyes were 151.4, 205.6, and 543.5 µg/mL, respectively, and the maximum ceftazidime aqueous humor concentrations are 64.6, 260.0, and 1176.3 µg/mL, respectively. There was no change in ECD, and ERG was not declined after intravitreal injection in all groups. Conclusions: The half-lives of vancomycin and ceftazidime in the aqueous humor were shorter in silicone oil-filled eyes than in normal and vitrectomized eyes. High antibiotic concentrations in silicone oil-filled eyes seemed to be well tolerated. Translational Relevance: This study aids in estimating how often an antibiotic should be intravitreally injected for endophthalmitis of silicone oil-filled eyes.

Clinical features of irreversible rejection after allogeneic uterus transplantation in cynomolgus macaques.

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.

First Successful Delivery after Uterus Transplantation in MHC-Defined 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.