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Anthony M Mancinelli,1 Jonathan M Vichich,1 Alexandra D Zinnen,1 Anna Marie Hugon,2 Viktoriya Bondarenko,1 Jeanette M Metzger,1 Heather A Simmons,1 Thaddeus G Golos,1,3,4 Marina E Emborg1,5 1Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI, USA; 2Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI, USA; 3Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI, USA; 4Department of Obstetrics and Gynecology, University of Wisconsin-Madison, Madison, WI, USA; 5Department of Medical Physics, University of Wisconsin-Madison, Madison, WI, USACorrespondence: Marina E Emborg Email emborg@primate.wisc.eduIntroduction: Gastrointestinal (GI) inflammation elicited by environmental factors is proposed to trigger Parkinson’s disease (PD) by stimulating accumulation of pathological α-synuclein (α-syn) in the enteric nervous system (ENS), which then propagates to the central nervous system via the vagus nerve. The goal of this study was to model, in nonhuman primates, an acute exposure to a common food-borne pathogen in order to assess whether the related acute GI inflammation could initiate persistent α-syn pathology in the ENS, ultimately leading to PD.Methods: Adult female cynomolgus macaques were inoculated by oral gavage with 1× 108 colony-forming units (CFUs) Listeria monocytogenes (LM, n=10) or vehicle (mock, n=3) and euthanized 2 weeks later. Evaluations included clinical monitoring, blood and fecal shedding of LM, and postmortem pathological analysis of colonic and cecal tissues.Results: LM inoculation of healthy adult cynomolgus macaques induced minimal to mild clinical signs of infection; LM shedding in feces was not seen in any of the animals nor was bacteremia detected. Colitis varied from none to moderate in LM-treated subjects and none to minimal in mock-treated subjects. Expression of inflammatory markers (HLA-DR, CD3, CD20), oxidative stress (8-OHDG), α-syn, and phosphorylated-α-syn in the enteric ganglia was not significantly different between treatment groups.Discussion: Our results demonstrate that cynomolgus macaques orally inoculated with LM present with a clinical response that resembles human LM exposure. They also suggest that acute exposure to food-borne pathogens is not sufficient to induce significant and persistent α-syn changes in healthy adult female subjects. Based on the results of this limited experimental setting, we propose that, if LM has a role in PD pathology, other underlying factors or conditions, such as male sex, inflammatory bowel disease, exposure to toxins, dysbiosis, and/or aging, are needed to be present.Keywords: Parkinson’s disease, alpha synuclein, nonhuman primate, listeria, inflammation, enteric nervous system

Nanoparticles offer promise as a mechanism to non-invasively deliver targeted placental therapeutics. Our previous studies utilizing intraplacental administration demonstrate efficient nanoparticle uptake into placental trophoblast cells and overexpression of human IGF1 (hIGF1). Nanoparticle-mediated placental overexpression of hIGF1 in small animal models of placental insufficiency and fetal growth restriction improved nutrient transport and restored fetal growth. The objective of this pilot study was to extend these studies to the pregnant nonhuman primate and develop a method for local delivery of nanoparticles to the placenta via maternal blood flow from the uterine artery. Nanoparticles containing hIGF1 plasmid driven by the placenta-specific PLAC1 promoter were delivered to a mid-gestation pregnant rhesus macaque via a catheterization approach that is clinically used for uterine artery embolization. Maternal-fetal interface, fetal and maternal tissues were collected four days post-treatment to evaluate the efficacy of hIGF1 treatment in the placenta. The uterine artery catheterization procedure and nanoparticle treatment was well tolerated by the dam and fetus through the four-day study period following catheterization. Nanoparticles were taken up by the placenta from maternal blood as plasmid-specific hIGF1 expression was detected in multiple regions of the placenta via in situ hybridization and qPCR. The uterine artery catheterization approach enabled successful delivery of nanoparticles to maternal circulation in close proximity to the placenta with no concerns to maternal or fetal health in this short-term feasibility study. In the future, this delivery approach can be used for preclinical evaluation of the long-term safety and efficacy of nanoparticle-mediated placental therapies in a rhesus macaque model., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Amniogenesis is triggered in a collection of pluripotent epiblast cells as the human embryo implants. To gain insights into the critical but poorly understood transcriptional machinery governing amnion fate determination, we examined the evolving transcriptome of a developing human pluripotent stem cell-derived amnion model at the single cell level. This analysis revealed several continuous amniotic fate progressing states with state-specific markers, which include a previously unrecognized CLDN10 + amnion progenitor state. Strikingly, we found that expression of CLDN10 is restricted to the amnion-epiblast boundary region in the human post-implantation amniotic sac model as well as in a peri-gastrula cynomolgus macaque embryo, bolstering the growing notion that, at this stage, the amnion-epiblast boundary is a site of active amniogenesis. Bioinformatic analysis of published primate peri-gastrula single cell sequencing data further confirmed that CLDN10 is expressed in cells progressing to amnion. Additionally, our loss of function analysis shows that CLDN10 promotes amniotic but suppresses primordial germ cell-like fate. Overall, this study presents a comprehensive amniogenic single cell transcriptomic resource and identifies a previously unrecognized CLDN10 + amnion progenitor population at the amnion-epiblast boundary of the primate peri-gastrula., Competing Interests: Competing interests: The authors declare no competing interest.

Background: Recently, dynamic contrast-enhanced (DCE) MRI with ferumoxytol as contrast agent has recently been introduced for the noninvasive assessment of placental structure and function throughout. However, it has not been demonstrated under pathological conditions., Purpose: To measure cotyledon-specific rhesus macaque maternal placental blood flow using ferumoxytol DCE MRI in a novel animal model for local placental injury., Study Type: Prospective animal model., Subjects: Placental injections of Tisseel (three with 0.5 mL and two with 1.5 mL), monocyte chemoattractant protein 1 (three with 100 μg), and three with saline as controls were performed in a total of 11 rhesus macaque pregnancies at approximate gestational day (GD 101). DCE MRI scans were performed prior (GD 100) and after (GD 115 and GD 145) the injection (term = GD 165)., Field Strength/sequence: 3 T, T1-weighted spoiled gradient echo sequence (product sequence, DISCO)., Assessment: Source images were inspected for motion artefacts from the mother or fetus. Placenta segmentation and DCE processing were performed for the dynamic image series to measure cotyledon specific volume, flow, and normalized flow. Overall placental histopathology was conducted for controls, Tisseel, and MCP-1 animals and regions of tissue infarctions and necrosis were documented. Visual inspections for potential necrotic tissue were conducted for the two Tisseelx3 animals., Statistical Tests: Wilcoxon rank sum test, significance level P < 0.05., Results: No motion artefacts were observed. For the group treated with 1.5 mL of Tisseel, significantly lower cotyledon volume, flow, and normalized flow per cotyledon were observed for the third gestational time point of imaging (day ~145), with mean normalized flow of 0.53 minute -1 . Preliminary histopathological analysis shows areas of tissue necrosis from a selected cotyledon in one Tisseel-treated (single dose) animal and both Tisseelx3 (triple dose) animals., Data Conclusion: This study demonstrates the feasibility of cotyledon-specific functional analysis at multiple gestational time points and injury detection in a placental rhesus macaque model through ferumoxytol-enhanced DCE MRI., Level of Evidence: NA TECHNICAL EFFICACY: Stage 2., (© 2024 The Authors. Journal of Magnetic Resonance Imaging published by Wiley Periodicals LLC on behalf of International Society for Magnetic Resonance in Medicine.)

Amniogenesis, a process critical for continuation of healthy pregnancy, is triggered in a collection of pluripotent epiblast cells as the human embryo implants. Previous studies have established that bone morphogenetic protein (BMP) signaling is a major driver of this lineage specifying process, but the downstream BMP-dependent transcriptional networks that lead to successful amniogenesis remain to be identified. This is, in part, due to the current lack of a robust and reproducible model system that enables mechanistic investigations exclusively into amniogenesis. Here, we developed an improved model of early amnion specification, using a human pluripotent stem cell-based platform in which the activation of BMP signaling is controlled and synchronous. Uniform amniogenesis is seen within 48 hr after BMP activation, and the resulting cells share transcriptomic characteristics with amnion cells of a gastrulating human embryo. Using detailed time-course transcriptomic analyses, we established a previously uncharacterized BMP-dependent amniotic transcriptional cascade, and identified markers that represent five distinct stages of amnion fate specification; the expression of selected markers was validated in early post-implantation macaque embryos. Moreover, a cohort of factors that could potentially control specific stages of amniogenesis was identified, including the transcription factor TFAP2A. Functionally, we determined that, once amniogenesis is triggered by the BMP pathway, TFAP2A controls the progression of amniogenesis. This work presents a temporally resolved transcriptomic resource for several previously uncharacterized amniogenesis states and demonstrates a critical intermediate role for TFAP2A during amnion fate specification., Competing Interests: NS, JW, AC, LJ, LT, XM, SR, JS, TG, CL, KT No competing interests declared, (© 2023, Sekulovski et al.)

Infection with clade I Mpox virus (MPXV) results in adverse pregnancy outcomes, yet the potential for vertical transmission resulting in fetal harm with clade IIb MPXV, the clade that is currently circulating in the Western Hemisphere, remains unknown. We established a rhesus macaque model of vertical MPXV transmission with early gestation inoculation. Three pregnant rhesus macaques were inoculated intradermally with 1.5 × 10^5 plaque forming units (PFU) of clade IIb MPXV near gestational day (GD) 30 and animals were monitored for viremia and maternal and fetal well-being. Animals were euthanized to collect tissues at 5, 14, or 25 days post-inoculation (dpi). Tissues were evaluated for viral DNA (vDNA) loads, infectious virus titers, histopathology, MPXV mRNA and protein localization, as well as MPXV protein co-localization with placental cells including, Hofbauer cells, mesenchymal stromal cells, endothelial cells, and trophoblasts. vDNA was detected in maternal blood and skin lesions by 5 dpi. Lack of fetal heartbeat was observed at 14 or 25 dpi for two dams indicating fetal demise; the third dam developed significant vaginal bleeding at 5 dpi and was deemed an impending miscarriage. vDNA was detected in placental and fetal tissue in both fetal demise cases. MPXV localized to placental villi by ISH and IHC. Clade IIb MPXV infection in pregnant rhesus macaques results in vertical transmission to the fetus and adverse pregnancy outcomes, like clade I MPXV. Further studies are needed to determine whether antiviral therapy with tecovirimat will prevent vertical transmission and improve pregnancy outcomes., One Sentence Summary: Clade IIb Mpox virus infection of pregnant rhesus macaques results in vertical transmission from mother to fetus and adverse pregnancy outcomes.

Zika virus (ZIKV) can be vertically transmitted during pregnancy resulting in a range of adverse pregnancy outcomes. The decidua is commonly found to be infected by ZIKV, yet the acute immune response to infection remains understudied in vivo . We hypothesized that in vivo African-lineage ZIKV infection induces a pro-inflammatory response in the decidua. To test this hypothesis, we evaluated the decidua in pregnant rhesus macaques within the first two weeks following infection with an African-lineage ZIKV and compared our findings to gestationally aged-matched controls. Decidual leukocytes were phenotypically evaluated using spectral flow cytometry, and cytokines and chemokines were measured in tissue homogenates from the decidua, placenta, and fetal membranes. The results of this study did not support our hypothesis. Although ZIKV RNA was detected in the decidual tissue samples from all ZIKV infected dams, phenotypic changes in decidual leukocytes and differences in cytokine profiles suggest that the decidua undergoes mild anti-inflammatory changes in response to that infection. Our findings emphasize the immunological state of the gravid uterus as a relatively immune privileged site that prioritizes tolerance of the fetus over mounting a pro-inflammatory response to clear infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Koenig, Vazquez, Leyva Jaimes, Mitzey, Stanic and Golos.)

The decidual immunome is dynamic, dramatically changing its composition across gestation. Early pregnancy is dominated by decidual NK cells, with a shift towards T cells later in pregnancy. However, the degree, timing, and subset-specific nature of leukocyte traffic between the decidua and systemic circulation during gestation remains poorly understood. Herein, we employed intravascular staining in pregnant C57BL/6J mice and cynomolgus macaques ( Macaca fascicularis ) to examine leukocyte traffic into the decidual basalis during pregnancy. Timed-mated or virgin mice were tail-vein injected with labelled αCD45 antibodies 24 hours and 5 minutes before sacrifice. Pregnant cynomolgus macaques (GD155) were infused with labelled αCD45 at 2 hours or 5 mins before necropsy. Decidual cells were isolated and resulting suspensions analyzed by flow cytometry. We found that the proportion of intravascular (IVAs)-negative leukocytes (cells labeled by the 24h infusion of αCD45 or unlabeled) decreased across murine gestation while recent immigrants (24h label only) increased in mid- to late-gestation. In the cynomolgus model our data confirmed differential labeling of decidual leukocytes by the infused antibody, with the 5 min infused animal having a higher proportion of IVAs+ cells compared to the 2hr infused animal. Decidual tissue sections from both macaques showed the presence of intravascularly labeled cells, either in proximity to blood vessels (5min infused animal) or deeper into decidual stroma (2hr infused animal). These results demonstrate the value of serial intravascular staining as a sensitive tool for defining decidual leukocyte traffic during pregnancy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Vazquez, Mohamed, Banerjee, Keding, Koenig, Leyva Jaimes, Fisher, Bove, Golos and Stanic.)

Embryo morphokinetic analysis through time-lapse embryo imaging is envisioned as a method to improve selection of developmentally competent embryos. Morphokinetic analysis could be utilized to evaluate the effects of experimental manipulation on pre-implantation embryo development. The objectives of this study were to establish a normative morphokinetic database for in vitro fertilized rhesus macaque embryos and to assess the impact of atypical initial cleavage patterns on subsequent embryo development and formation of embryo outgrowths. The cleavage pattern and the timing of embryo developmental events were annotated retrospectively for unmanipulated in vitro fertilized rhesus macaque blastocysts produced over four breeding seasons. Approximately 50% of the blastocysts analyzed had an abnormal early cleavage event. The time to the initiation of embryo compaction and the time to completion of hatching was significantly delayed in blastocysts with an abnormal early cleavage event compared to blastocysts that had cleaved normally. Embryo hatching, attachment to an extracellular matrix, and growth during the implantation stage in vitro was not impacted by the initial cleavage pattern. These data establish normative morphokinetic parameters for in vitro fertilized rhesus macaque embryos and suggest that cleavage anomalies may not impact embryo implantation rates following embryo transfer., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: The bacterium Listeria monocytogenes (Lm) is associated with adverse pregnancy outcomes. Infection occurs through consumption of contaminated food that is disseminated to the maternal-fetal interface. The influence on the gastrointestinal microbiome during Lm infection remains unexplored in pregnancy. The objective of this study was to determine the impact of listeriosis on the gut microbiota of pregnant macaques., Methods: A non-human primate model of listeriosis in pregnancy has been previously described. Both pregnant and non-pregnant cynomolgus macaques were inoculated with Lm and bacteremia and fecal shedding were monitored for 14 days. Non-pregnant animal tissues were collected at necropsy to determine bacterial burden, and fecal samples from both pregnant and non-pregnant animals were evaluated by 16S rRNA next-generation sequencing., Results: Unlike pregnant macaques, non-pregnant macaques did not exhibit bacteremia, fecal shedding, or tissue colonization by Lm. Dispersion of Lm during pregnancy was associated with a significant decrease in alpha diversity of the host gut microbiome, compared to non-pregnant counterparts. The combined effects of pregnancy and listeriosis were associated with a significant loss in microbial richness, although there were increases in some genera and decreases in others., Conclusions: Although pregnancy alone is not associated with gut microbiome disruption, we observed dysbiosis with listeriosis during pregnancy. The macaque model may provide an understanding of the roles that pregnancy and the gut microbiota play in the ability of Lm to establish intestinal infection and disseminate throughout the host, thereby contributing to adverse pregnancy outcomes and risk to the developing fetus., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Zika virus (ZIKV) infection during pregnancy results in a spectrum of birth defects and neurodevelopmental deficits in prenatally exposed infants, with no clear understanding of why some pregnancies are more severely affected. Differential control of maternal ZIKV infection may explain the spectrum of adverse outcomes., Methods: Here, we investigated whether the magnitude and breadth of the maternal ZIKV-specific antibody response is associated with better virologic control using a rhesus macaque model of prenatal ZIKV infection. We inoculated 18 dams with an Asian-lineage ZIKV isolate (PRVABC59) at 30-45 gestational days. Plasma vRNA and infectious virus kinetics were determined over the course of pregnancy, as well as vRNA burden in the maternal-fetal interface (MFI) at delivery. Binding and neutralizing antibody assays were performed to determine the magnitude of the ZIKV-specific IgM and IgG antibody responses throughout pregnancy, along with peptide microarray assays to define the breadth of linear ZIKV epitopes recognized., Results: Dams with better virologic control (n= 9) cleared detectable infectious virus and vRNA from the plasma by 7 days post-infection (DPI) and had a lower vRNA burden in the MFI at delivery. In comparison, dams with worse virologic control (n= 9) still cleared detectable infectious virus from the plasma by 7 DPI but had vRNA that persisted longer, and had higher vRNA burden in the MFI at delivery. The magnitudes of the ZIKV-specific antibody responses were significantly lower in the dams with better virologic control, suggesting that higher antibody titers are not associated with better control of ZIKV infection. Additionally, the breadth of the ZIKV linear epitopes recognized did not differ between the dams with better and worse control of ZIKV infection., Discussion: Thus, the magnitude and breadth of the maternal antibody responses do not seem to impact maternal virologic control. This may be because control of maternal infection is determined in the first 7 DPI, when detectable infectious virus is present and before robust antibody responses are generated. However, the presence of higher ZIKV-specific antibody titers in dams with worse virologic control suggests that these could be used as a biomarker of poor maternal control of infection and should be explored further., Competing Interests: Authors ES and JT are employed by the company Nimble Therapeutics, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Krabbe, Razo, Abraham, Spanton, Shi, Bhattacharya, Bohm, Pritchard, Weiler, Mitzey, Eickhoff, Sullivan, Tan, Aliota, Friedrich, O’Connor, Golos and Mohr.)

Zika virus (ZIKV) can be transmitted vertically from mother to fetus during pregnancy, resulting in a range of outcomes including severe birth defects and fetal/infant death. Potential pathways of vertical transmission in utero have been proposed but remain undefined. Identifying the timing and routes of vertical transmission of ZIKV may help us identify when interventions would be most effective. Furthermore, understanding what barriers ZIKV overcomes to effect vertical transmission may help improve models for evaluating infection by other pathogens during pregnancy. To determine the pathways of vertical transmission, we inoculated 12 pregnant rhesus macaques with an African-lineage ZIKV at gestational day 30 (term is 165 days). Eight pregnancies were surgically terminated at either seven or 14 days post-maternal infection. Maternal-fetal interface and fetal tissues and fluids were collected and evaluated for ZIKV using RT-qPCR, in situ hybridization, immunohistochemistry, and plaque assays. Four additional pregnant macaques were inoculated and terminally perfused with 4% paraformaldehyde at three, six, nine, or ten days post-maternal inoculation. For these four cases, the entire fixed pregnant uterus was evaluated with in situ hybridization for ZIKV RNA. We determined that ZIKV can reach the MFI by six days after infection and infect the fetus by ten days. Infection of the chorionic membrane and the extraembryonic coelomic fluid preceded infection of the fetus and the mesenchymal tissue of the placental villi. We did not find evidence to support a transplacental route of ZIKV vertical transmission via infection of syncytiotrophoblasts or villous cytotrophoblasts. The pattern of infection observed in the maternal-fetal interface provides evidence of paraplacental vertical ZIKV transmission through the chorionic membrane, the outer layer of the fetal membranes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

The microbiome has been shown, or implicated to be involved, in multiple facets of human health and disease, including not only gastrointestinal health but also metabolism, immunity, and neurology. Although the predominant focus of microbiome research has been on the gut, other microbial communities such as the vaginal or cervical microbiome are likely involved in physiological homeostasis. Emerging studies also aim to understand the role of different microbial niches, such as the endometrial or placental microbial communities, on the physiology and pathophysiology of reproduction, including their impact on reproductive success and the etiology of adverse pregnancy outcomes (APOs). The study of the microbiome during pregnancy, specifically how changes in maternal microbial communities can lead to dysfunction and disease, can advance the understanding of reproductive health and the etiology of APOs. In this review, we will discuss the current state of non-human primate (NHP) reproductive microbiome research, highlight the progress with NHP models of reproduction, and the diagnostic potential of microbial alterations in a clinical setting to promote pregnancy health. NHP reproductive biology studies have the potential to expand the knowledge and understanding of female reproductive tract microbial communities and host-microbe or microbe-microbe interactions associated with reproductive health through sequencing and analysis. Furthermore, in this review, we aim to demonstrate that macaques are uniquely suited as high-fidelity models of human female reproductive pathology., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Congenital Zika virus (ZIKV) infection can result in birth defects, including malformations in the fetal brain and visual system. There are two distinct genetic lineages of ZIKV: African and Asian. Asian-lineage ZIKVs have been associated with adverse pregnancy outcomes in humans; however, recent evidence from experimental models suggests that African-lineage viruses can also be vertically transmitted and cause fetal harm., Methodology/principal Findings: To evaluate the pathway of vertical transmission of African-lineage ZIKV, we inoculated nine pregnant rhesus macaques (Macaca mulatta) subcutaneously with 44 plaque-forming units of a ZIKV strain from Senegal, (ZIKV-DAK). Dams were inoculated either at gestational day 30 or 45. Following maternal inoculation, pregnancies were surgically terminated seven or 14 days later and fetal and maternal-fetal interface tissues were collected and evaluated. Infection in the dams was evaluated via plasma viremia and neutralizing antibody titers pre- and post- ZIKV inoculation. All dams became productively infected and developed strong neutralizing antibody responses. ZIKV RNA was detected in maternal-fetal interface tissues (placenta, decidua, and fetal membranes) by RT-qPCR and in situ hybridization. In situ hybridization detected ZIKV predominantly in the decidua and revealed that the fetal membranes may play a role in ZIKV vertical transmission. Infectious ZIKV was detected in the amniotic fluid of three pregnancies and one fetus had ZIKV RNA detected in multiple tissues. No significant pathology was observed in any fetus; and ZIKV did not have a substantial effect on the placenta., Conclusions/significance: This study demonstrates that a very low dose of African-lineage ZIKV can be vertically transmitted to the macaque fetus during pregnancy. The low inoculating dose used in this study suggests a low minimal infectious dose for rhesus macaques. Vertical transmission with a low dose in macaques further supports the high epidemic potential of African ZIKV strains., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Background: Understanding gait development is essential for identifying motor impairments in neurodevelopmental disorders. Defining typical gait development in a rhesus macaque model is critical prior to characterizing abnormal gait. The goal of this study was to 1) explore the feasibility of using the Noldus Catwalk to assess gait in infant rhesus macaques and 2) provide preliminary normative data of gait development during the first month of life., New Method: The Noldus Catwalk was used to assess gait speed, dynamic and static paw measurements, and interlimb coordination in twelve infant rhesus macaques at 14, 21, and 28 days of age. All macaque runs were labeled as a diagonal or non-diagonal walking pattern., Results: Infant rhesus macaques primarily used a diagonal (mature) walking pattern as early as 14 days of life. Ten infant rhesus macaques (83.3%) were able to successfully walk across the Noldus Catwalk at 28 days of life. Limited differences in gait parameters were observed between timepoints because of the variability within the group at 14, 21, and 28 days., Comparison With Existing Methods: No prior gait analysis system has been used to provide objective quantification of gait parameters for infant macaques., Conclusions: The Catwalk system can be utilized to quantify gait in infant rhesus macaques less than 28 days old. Future applications to infant rhesus macaques could provide a better understanding of gait development and early differences within various neurodevelopmental disorders., Competing Interests: Declaration of Competing Interest No conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Introduction: Genome editing by CRISPR-Cas9 approaches offers promise for introducing or correcting disease-associated mutations for research and clinical applications. Nonhuman primates are physiologically closer to humans than other laboratory animal models, providing ideal candidates for introducing human disease-associated mutations to develop models of human disease. The incidence of large chromosomal anomalies in CRISPR-Cas9-edited human embryos and cells warrants comprehensive genotypic investigation of editing outcomes in primate embryos. Our objective was to evaluate on- and off-target editing outcomes in CCR5 CRISPR-Cas9-targeted Mauritian cynomolgus macaque embryos. Methods: DNA isolated from individual blastomeres of two embryos, along with paternal and maternal DNA, was subjected to whole genome sequencing (WGS) analysis. Results: Large deletions were identified in macaque blastomeres at the on-target site that were not previously detected using PCR-based methods. De novo mutations were also identified at predicted CRISPR-Cas9 off-target sites. Discussion: This is the first report of WGS analysis of CRISPR-Cas9-targeted nonhuman primate embryonic cells, in which a high editing efficiency was coupled with the incidence of editing errors in cells from two embryos. These data demonstrate that comprehensive sequencing-based methods are warranted for evaluating editing outcomes in primate embryos, as well as any resultant offspring to ensure that the observed phenotype is due to the targeted edit and not due to unidentified off-target mutations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Schmidt, Kim, Strelchenko, Gierczic, Pavelec, Golos and Slukvin.)

Identification of placental dysfunction in early pregnancy with noninvasive imaging could be a valuable tool for assessing maternal and fetal risk. Dynamic contrast enhanced (DCE) magnetic resonance imaging (MRI) can be a powerful tool for interrogating placenta health. After inoculation with Zika virus or sham inoculation at gestation age (GA) 45 or 55 days, animals were imaged up to three times at GA65, GA100, and GA145. DCE MRI images were acquired at all imaging sessions using ferumoxytol, an iron nanoparticle-based contrast agent, and analyzed for placental intervillous blood flow, number of perfusion domains, and perfusion domain volume. Cesarean section was performed at GA155, and the placenta was photographed and dissected for histopathology. Photographs were used to align cotyledons with estimated perfusion domains from MRI, allowing comparison of estimated cotyledon volume to pathology. Monkeys were separated into high and low pathology groups based on the average number of pathologies present in the placenta. Perfusion domain flow, volume, and number increased through gestation, and total blood flow increased with gestation for both low pathology and high pathology groups. A statistically significant decrease in perfusion domain volume associated with pathology was detected at all gestational ages. Individual perfusion domain flow comparisons demonstrated a statistically significant decrease with pathology at GA100 and GA145, but not GA65. Since ferumoxytol is currently used to treat anemia during human pregnancy and as an off-label MRI contrast agent, future transition of this work to human pregnancy may be possible., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Nonhuman primates (NHPs) represent one of the most important models for preclinical studies of novel biomedical interventions. In contrast with small animal models, however, widespread utilization of NHPs is restricted by cost, logistics, and availability. Therefore, we sought to develop a translational primatized mouse model, akin to a humanized mouse, to allow for high-throughput in vivo experimentation leveraged to inform large animal immunology-based studies. We found that adult rhesus macaque mobilized blood (AMb) CD34+-enriched hematopoietic stem and progenitor cells (HSPCs) engrafted at low but persistent levels in immune-deficient mice harboring transgenes for human (NHP cross-reactive) GM-CSF and IL3, but did not in mice with wild-type murine cytokines lacking NHP cross-reactivity. To enhance engraftment, fetal liver-derived HSPCs were selected as the infusion product based on an increased CD34 hi fraction compared with AMb and bone marrow. Coupled with cotransplantation of rhesus fetal thymic fragments beneath the mouse kidney capsule, fetal liver-derived HSPC infusion in cytokine-transgenic mice yielded robust multilineage lymphohematopoietic engraftment. The emergent immune system recapitulated that of the fetal monkey, with similar relative frequencies of lymphocyte, granulocyte, and monocyte subsets within the thymic, secondary lymphoid, and peripheral compartments. Importantly, while exhibiting a predominantly naïve phenotype, in vitro functional assays demonstrated robust cellular activation in response to nonspecific and allogenic stimuli. This primatized mouse represents a viable and translatable model for the study of hematopoietic stem cell physiology, immune development, and functional immunology in NHPs. Summary Sentence: Engraftment of rhesus macaque hematopoietic tissues in immune-deficient mice yields a robust BLT/NeoThy-type primatized mouse model for studying nonhuman primate hematopoiesis and immune function in vivo., (©2022 Society for Leukocyte Biology.)

Nonhuman primates (NHPs) are well-established basic and translational research models for human immunodeficiency virus (HIV) infections and pathophysiology, hematopoietic stem cell (HSC) transplantation, and assisted reproductive technologies. Recent advances in CRISPR/Cas9 gene editing technologies present opportunities to refine NHP HIV models for investigating genetic factors that affect HIV replication and designing cellular therapies that exploit genetic barriers to HIV infections, including engineering mutations into CCR5 and conferring resistance to HIV/simian immunodeficiency virus (SIV) infections. In this report, we provide an overview of recent advances and challenges in gene editing NHP embryos and discuss the value of genetically engineered animal models for developing novel stem cell-based therapies for curing HIV., (© 2022. The Author(s).)

Countermeasures against Zika virus (ZIKV), including vaccines, are frequently tested in nonhuman primates (NHP). Macaque models are important for understanding how ZIKV infections impact human pregnancy due to similarities in placental development. The lack of consistent adverse pregnancy outcomes in ZIKV-affected pregnancies poses a challenge in macaque studies where group sizes are often small (4-8 animals). Studies in small animal models suggest that African-lineage Zika viruses can cause more frequent and severe fetal outcomes. No adverse outcomes were observed in macaques exposed to 1x104 PFU (low dose) of African-lineage ZIKV at gestational day (GD) 45. Here, we exposed eight pregnant rhesus macaques to 1x108 PFU (high dose) of African-lineage ZIKV at GD 45 to test the hypothesis that adverse pregnancy outcomes are dose-dependent. Three of eight pregnancies ended prematurely with fetal death. ZIKV was detected in both fetal and placental tissues from all cases of early fetal loss. Further refinements of this exposure system (e.g., varying the dose and timing of infection) could lead to an even more consistent, unambiguous fetal loss phenotype for assessing ZIKV countermeasures in pregnancy. These data demonstrate that high-dose exposure to African-lineage ZIKV causes pregnancy loss in macaques and also suggest that ZIKV-induced first trimester pregnancy loss could be strain-specific., Competing Interests: The authors have declared that no competing interests exist.

Allogeneic hematopoietic stem cell transplants (allo-HSCTs) dramatically reduce HIV reservoirs in antiretroviral therapy (ART) suppressed individuals. However, the mechanism(s) responsible for these post-transplant viral reservoir declines are not fully understood. Therefore, we modeled allo-HSCT in ART-suppressed simian-human immunodeficiency virus (SHIV)-infected Mauritian cynomolgus macaques (MCMs) to illuminate factors contributing to transplant-induced viral reservoir decay. Thus, we infected four MCMs with CCR5-tropic SHIV162P3 and started them on ART 6-16 weeks post-infection (p.i.), maintaining continuous ART during myeloablative conditioning. To prevent graft-versus-host disease (GvHD), we transplanted allogeneic MHC-matched α/β T cell-depleted bone marrow cells and prophylactically treated the MCMs with cyclophosphamide and tacrolimus. The transplants produced ~ 85% whole blood donor chimerism without causing high-grade GvHD. Consequently, three MCMs had undetectable SHIV DNA in their blood post-transplant. However, SHIV-harboring cells persisted in various tissues, with detectable viral DNA in lymph nodes and tissues between 38 and 62 days post-transplant. Further, removing one MCM from ART at 63 days post-transplant resulted in SHIV rapidly rebounding within 7 days of treatment withdrawal. In conclusion, transplanting SHIV-infected MCMs with allogeneic MHC-matched α/β T cell-depleted bone marrow cells prevented high-grade GvHD and decreased SHIV-harboring cells in the blood post-transplant but did not eliminate viral reservoirs in tissues., (© 2022. The Author(s).)

There are currently no approved drugs to treat Zika virus (ZIKV) infection during pregnancy. Hyperimmune globulin products such as VARIZIG and WinRho are FDA-approved to treat conditions during pregnancy such as Varicella Zoster virus infection and Rh-incompatibility. We administered ZIKV-specific human immune globulin as a treatment in pregnant rhesus macaques one day after subcutaneous ZIKV infection. All animals controlled ZIKV viremia following the treatment and generated robust levels of anti-Zika virus antibodies in their blood. No adverse fetal or infant outcomes were identified in the treated animals, yet the placebo control treated animals also did not have signs related to congenital Zika syndrome (CZS). Human immune globulin may be a viable prophylaxis and treatment option for ZIKV infection during pregnancy, however, more studies are required to fully assess the impact of this treatment to prevent CZS., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: DHO is a paid consultant for Battelle, devoted to research in the areas of assisting in the design and interpretation of their nonhuman primate ZIKV studies. His relationship does not carry with it any restrictions on publication, and any associated intellectual property will be disclosed and processed according to UW-Madison policy. None of the animals used in this study are involved in any studies with Battelle. The publication’s contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. The following patents have been filed that pertain to the results in this manuscript: PCT/IB2019/055275 and PCT/IB2019/053463.

Zika virus (ZIKV) infection at the maternal-placental interface is associated with adverse pregnancy outcomes including fetal demise and pregnancy loss. To determine how infection impacts placental trophoblasts, we utilized rhesus macaque trophoblast stem cells (TSC) that can be differentiated into early gestation syncytiotrophoblasts (ST) and extravillous trophoblasts (EVT). TSCs and STs, but not EVTs, were highly permissive to productive infection with ZIKV strain DAK AR 41524. The impact of ZIKV on the cellular transcriptome showed that infection of TSCs and STs increased expression of immune related genes, including those involved in type I and type III interferon responses. ZIKV exposure altered extracellular vesicle (EV) mRNA, miRNA and protein cargo, including ZIKV proteins, regardless of productive infection. These findings suggest that early gestation macaque TSCs and STs are permissive to ZIKV infection, and that EV analysis may provide a foundation for identifying non-invasive biomarkers of placental infection in a highly translational model., (© 2022. The Author(s).)

Infants exposed to Zika virus (ZIKV) prenatally may develop birth defects, developmental deficits, or remain asymptomatic. It is unclear why some infants are more affected than others, although enhancement of maternal ZIKV infection via immunity to an antigenically similar virus, dengue virus (DENV), may play a role. We hypothesized that DENV immunity may worsen prenatal ZIKV infection and developmental deficits in offspring. We utilized a translational macaque model to examine how maternal DENV immunity influences ZIKV-exposed infant macaque neurodevelopment in the first month of life. We inoculated eight macaques with prior DENV infection with ZIKV, five macaques with ZIKV, and four macaques with saline. DENV/ZIKV-exposed infants had significantly worse visual orientation skills than ZIKV-exposed infants whose mothers were DENV-naive, with no differences in motor, sensory or state control development. ZIKV infection characteristics and pregnancy outcomes did not individually differ between dams with and without DENV immunity, but when multiple factors were combined in a multivariate model, maternal DENV immunity combined with ZIKV infection characteristics and pregnancy parameters predicted select developmental outcomes. We demonstrate that maternal DENV immunity exacerbates visual orientation and tracking deficits in ZIKV-exposed infant macaques, suggesting that human studies should evaluate how maternal DENV immunity impacts long-term neurodevelopment.

Concerns have arisen that pre-existing immunity to dengue virus (DENV) could enhance Zika virus (ZIKV) disease, due to the homology between ZIKV and DENV and the observation of antibody-dependent enhancement (ADE) among DENV serotypes. To date, no study has examined the impact of pre-existing DENV immunity on ZIKV pathogenesis during pregnancy in a translational non-human primate model. Here we show that macaques with a prior DENV-2 exposure had a higher burden of ZIKV vRNA in maternal-fetal interface tissues as compared to DENV-naive macaques. However, pre-existing DENV immunity had no detectable impact on ZIKV replication kinetics in maternal plasma, and all pregnancies progressed to term without adverse outcomes or gross fetal abnormalities detectable at delivery. Understanding the risks of ADE to pregnant women worldwide is critical as vaccines against DENV and ZIKV are developed and licensed and as DENV and ZIKV continue to circulate., Competing Interests: The authors have declared that no competing interests exist.

Following the Zika virus (ZIKV) outbreak in the Americas, ZIKV was causally associated with microcephaly and a range of neurological and developmental symptoms, termed congenital Zika syndrome (CZS). The viruses responsible for this outbreak belonged to the Asian lineage of ZIKV. However, in vitro and in vivo studies assessing the pathogenesis of African-lineage ZIKV demonstrated that African-lineage isolates often replicated to high titers and caused more-severe pathology than Asian-lineage isolates. To date, the pathogenesis of African-lineage ZIKV in a translational model, particularly during pregnancy, has not been rigorously characterized. Here, we infected four pregnant rhesus macaques with a low-passage-number strain of African-lineage ZIKV and compared its pathogenesis to those for a cohort of four pregnant rhesus macaques infected with an Asian-lineage isolate and a cohort of mock-inoculated controls. The viral replication kinetics for the two experimental groups were not significantly different, and both groups developed robust neutralizing antibody titers above levels considered to be protective. There was no evidence of significant fetal head growth restriction or gross fetal harm at delivery (1 to 1.5 weeks prior to full term) in either group. However, a significantly higher burden of ZIKV viral RNA (vRNA) was found in the maternal-fetal interface tissues of the macaques exposed to an African-lineage isolate. Our findings suggest that ZIKV of any genetic lineage poses a threat to pregnant individuals and their infants. IMPORTANCE ZIKV was first identified in 1947 in Africa, but most of our knowledge of ZIKV is based on studies of the distinct Asian genetic lineage, which caused the outbreak in the Americas in 2015 to 2016. In its most recent update, the WHO stated that improved understanding of African-lineage ZIKV pathogenesis during pregnancy must be a priority. The recent detection of African-lineage isolates in Brazil underscores the need to understand the impact of these viruses. Here, we provide the first comprehensive assessment of African-lineage ZIKV infection during pregnancy in a translational nonhuman primate model. We show that African-lineage isolates replicate with kinetics similar to those of Asian-lineage isolates and can infect the placenta. However, there was no evidence of more-severe outcomes with African-lineage isolates. Our results highlight both the threat that African-lineage ZIKV poses to pregnant individuals and their infants and the need for epidemiological and translational in vivo studies with African-lineage ZIKV.

A wide array of pathogens has the potential to injure the fetus and induce teratogenesis, the process by which mutations in fetal somatic cells lead to congenital malformations. Rubella virus was the first infectious disease to be linked to congenital malformations due to an infection in pregnancy, which can include congenital cataracts, microcephaly, hearing impairment and congenital heart disease. Currently, human cytomegalovirus (HCMV) is the leading infectious cause of congenital malformations globally, affecting 1 in every 200 infants. However, our knowledge of teratogenic viruses and pathogens is far from complete. New emerging infectious diseases may induce teratogenesis, similar to Zika virus (ZIKV) that caused a global pandemic in 2016-2017; thousands of neonates were born with congenital microcephaly due to ZIKV exposure in utero , which also included a spectrum of injuries to the brain, eyes and spinal cord. In addition to congenital anomalies, permanent injury to fetal and neonatal organs, preterm birth, stillbirth and spontaneous abortion are known consequences of a broader group of infectious diseases including group B streptococcus (GBS), Listeria monocytogenes , Influenza A virus (IAV), and Human Immunodeficiency Virus (HIV). Animal models are crucial for determining the mechanism of how these various infectious diseases induce teratogenesis or organ injury, as well as testing novel therapeutics for fetal or neonatal protection. Other mammalian models differ in many respects from human pregnancy including placentation, labor physiology, reproductive tract anatomy, timeline of fetal development and reproductive toxicology. In contrast, non-human primates (NHP) most closely resemble human pregnancy and exhibit key similarities that make them ideal for research to discover the mechanisms of injury and for testing vaccines and therapeutics to prevent teratogenesis, fetal and neonatal injury and adverse pregnancy outcomes (e.g., stillbirth or spontaneous abortion). In this review, we emphasize key contributions of the NHP model pre-clinical research for ZIKV, HCMV, HIV, IAV, L. monocytogenes , Ureaplasma species, and GBS. This work represents the foundation for development and testing of preventative and therapeutic strategies to inhibit infectious injury of human fetuses and neonates., Competing Interests: SP is a consultant for Merck, Moderna, Pfizer, and Dynavax vaccines, and collaborates with Merck and Moderna on sponsored research programs around CMV vaccines. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li, Brokaw, Furuta, Coler, Obregon-Perko, Chahroudi, Wang, Permar, Hotchkiss, Golos, Rajagopal and Adams Waldorf.)

Background: Biomedical research has recently focused on developing new models of human disease by implementing genome-editing strategies in non-human primates (NHPs) to introduce relevant gene mutations. There is a need to establish objective semen evaluation methods to select sires for in vitro fertilization to perform germline editing in embryos., Methods: Sperm motility kinematic parameters were evaluated using a computer-assisted semen analysis (CASA) instrument for rhesus macaques (Macaca mulatta), cynomolgus macaques (Macaca fascicularis), and common marmosets (Callithrix jacchus)., Results: Normative sperm kinematic parameters were established, revealing differences between marmosets and macaques. The impact of season on rhesus macaque sperm motility was modest, where changes in sperm motility related to season were dependent on the individual male., Conclusions: These data provide a baseline of normative kinematic parameters for three captive NHP species, in which implementation of CASA may serve as a tool to evaluate NHP semen quality., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Monitoring the health of a pregnancy is of utmost importance to both the fetus and the mother. The diagnosis of pregnancy complications typically occurs after the manifestation of symptoms, and limited preventative measures or effective treatments are available. Traditionally, pregnancy health is evaluated by analyzing maternal serum hormone levels, genetic testing, ultrasonographic imaging, and monitoring maternal symptoms. However, researchers have reported a difference in extracellular vesicle (EV) quantity and cargo between healthy and at-risk pregnancies. Thus, placental EVs (PEVs) may help to understand normal and aberrant placental development, monitor pregnancy health in terms of developing placental pathologies, and assess the impact of environmental influences, such as infection, on pregnancy. The diagnostic potential of PEVs could allow for earlier detection of pregnancy complications via noninvasive sampling and frequent monitoring. Understanding how PEVs serve as a means of communication with maternal cells and recognizing their potential utility as a readout of placental health have sparked a growing interest in basic and translational research. However, to date, PEV research with animal models lags behind human studies. The strength of animal pregnancy models is that they can be used to assess placental pathologies in conjunction with isolation of PEVs from fluid samples at different time points throughout gestation. Assessing PEV cargo in animals within normal and complicated pregnancies will accelerate the translation of PEV analysis into the clinic for potential use in prognostics. We propose that appropriate animal models of human pregnancy complications must be established in the PEV field., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Nonhuman primates are excellent models for studying human placentation as experimental manipulations in vitro can be translated to in vivo pregnancy. Our objective was to develop macaque trophoblast stem cells (TSCs) as an in vitro platform for future assessment of primate trophoblast development and function. Macaque TSC lines were generated by isolating first and second trimester placental villous cytotrophoblasts followed by culture in TSC medium to maintain cellular proliferation. TSCs grew as mononuclear colonies, whereas upon induction of syncytiotrophoblast (ST) differentiation multinuclear structures appeared, indicative of syncytium formation. Chorionic gonadotropin secretion was > 4000-fold higher in ST culture media compared to TSC media. The secretion of chorionic gonadotropin by TSC-derived ST reflects a reprogramming of macaque TSCs to an earlier pregnancy phenotype. Characteristic trophoblast hallmarks were defined in TSCs and ST including expression of C19MC miRNAs and the macaque placental nonclassical MHC class I molecule, Mamu-AG. Extravillous trophoblasts (EVTs) were derived that express macaque EVT markers Mamu-AG and CD56, and also secrete high levels of MMP2. Our analyses of macaque TSCs suggests that these cells represent a proliferative, self-renewing population capable of differentiating to STs and EVTs in vitro thereby establishing an experimental model of primate placentation.

The objective of this study was to optimize cryopreservation of sperm from Mauritian cynomolgus macaques (MCM) in defined conditions. Sperm viability and motility were compared between sperm cryopreserved in chemically-defined freezing media with variable osmolarity and the presence of either ethylene glycol or glycerol. The highest percentage viability (after freeze-thaw) was seen in sperm samples that were cryopreserved in medium with an osmolarity of 310 mOsm, while higher osmolarities markedly decreased sperm viability. Ethylene glycol and glycerol at concentrations of 4.6% and 5%, respectively, preserved sperm viability to an equivalent degree. Although higher motility rates and higher straight-line velocities were observed in sperm samples frozen in glycerol compared with ethylene glycol, these differences were not statistically significant. Thawed sperm frozen in defined conditions with glycerol were capable of fertilizing MCM oocytes in vitro, with development to the blastocyst stage. The protocol described here provides an effective method for cryopreservation of sperm to facilitate subsequent in vitro fertilization and genome editing of embryos in MCM species.

Although sexual transmission of Zika virus (ZIKV) is well-documented, the viral reservoir(s) in the male reproductive tract remains uncertain in humans and immune-intact animal models. We evaluated the presence of ZIKV in a rhesus macaque pilot study to determine persistence in semen, assess the impact of infection on sperm functional characteristics, and define the viral reservoir in the male reproductive tract. Five adult male rhesus monkeys were inoculated with 105 PFU of Asian-lineage ZIKV isolate PRVABC59, and two males were inoculated with the same dose of African-lineage ZIKV DAKAR41524. Viremia and viral RNA (vRNA) shedding in semen were monitored, and a cohort of animals were necropsied for tissue collection to assess tissue vRNA burden and histopathology. All animals exhibited viremia for limited periods (1-11 days); duration of shedding did not differ significantly between viral isolates. There were sporadic low levels of vRNA in the semen from some, but not all animals. Viral RNA levels in reproductive tract tissues were also modest and present in the epididymis in three of five cases, one case in the vas deferens, but not detected in testis, seminal vesicles or prostate. ZIKV infection did not impact semen motility parameters as assessed by computer-assisted sperm analysis. Despite some evidence of prolonged ZIKV RNA shedding in human semen and high tropism of ZIKV for male reproductive tract tissues in mice deficient in Type 1 interferon signaling, in the rhesus macaques assessed in this pilot study, we did not consistently find ZIKV RNA in the male reproductive tract., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The discovery that CCR5 serves as an R5-HIV-1 co-receptor, coupled with findings of protection from HIV infection in individuals lacking CCR5, led to the exploration of novel therapeutic strategies for HIV infection based on genome editing of CCR5. Advancing translation of CCR5-mutant-based cellular therapies for HIV requires development of novel physiologically relevant animal models. Mauritian cynomolgus macaques (MCMs), with high degree of MHC allele sharing, are valuable models for HIV-1 research and stem cell therapies. To facilitate the generation of a CCR5-mutant MHC-defined MCM model, we explored editing the CCR5 gene in MCM embryos via CRISPR-Cas9. We refined ovarian stimulation and in vitro fertilization (IVF) methods established for Chinese cynomolgus macaques to generate in vitro MCM embryos. Time-lapse embryo imaging was performed to assess the timing of MCM embryonic developmental events in control and CRISPR-Cas9 microinjected embryos. Using a dual-guide gene targeting approach, biallelic deletions in the CCR5 gene were introduced into ~ 23-37% of MCM embryos. In addition, single blastomere PCR analysis revealed mosaicism in CCR5 editing within the same embryo. Successful development of IVF and CCR5 editing protocols in MCM embryos lays a foundation for the creation of CCR5-mutant MCMs to assess novel stem cell-based HIV therapeutics.

Congenital Zika virus (ZIKV) exposure results in a spectrum of disease ranging from severe birth defects to delayed onset neurodevelopmental deficits. ZIKV-related neuropathogenesis, predictors of birth defects, and neurodevelopmental deficits are not well defined in people. Here we assess the methodological and statistical feasibility of a congenital ZIKV exposure macaque model for identifying infant neurobehavior and brain abnormalities that may underlie neurodevelopmental deficits. We inoculated five pregnant macaques with ZIKV and mock-inoculated one macaque in the first trimester. Following birth, growth, ocular structure/function, brain structure, hearing, histopathology, and neurobehavior were quantitatively assessed during the first week of life. We identified the typical pregnancy outcomes of congenital ZIKV infection, with fetal demise and placental abnormalities. We estimated sample sizes needed to define differences between groups and demonstrated that future studies quantifying brain region volumes, retinal structure, hearing, and visual pathway function require a sample size of 14 animals per group (14 ZIKV, 14 control) to detect statistically significant differences in at least half of the infant exam parameters. Establishing the parameters for future studies of neurodevelopmental outcomes following congenital ZIKV exposure in macaques is essential for robust and rigorous experimental design., Competing Interests: DHO is a paid consultant for Battelle, devoted to research in the areas of assisting in the design and interpretation of their nonhuman primate ZIKV studies. His relationship does not carry with it any restrictions on publication, and any associated intellectual property will be disclosed and processed according to UW-Madison policy. None of the animals used in this study are involved in any studies with Battelle.

Zika virus (ZIKV) infection is associated with adverse pregnancy outcomes in humans, and infection in the first trimester can lead to miscarriage and stillbirth. Vertical and sexual transmissions of ZIKV have been demonstrated, yet the impact of infection during the initial stages of pregnancy remains unexplored. Here we defined the impact of ZIKV on early embryonic and placental development with a rhesus macaque model. During in vitro fertilization (IVF), macaque gametes were inoculated with a physiologically relevant dose of 5.48log10 plaque-forming units (PFU) of Zika virus/H.sapiens-tc/PUR/2015/PRVABC59&#95;v3c2. Exposure at fertilization did not alter blastocyst formation rates compared to controls. To determine the impact of ZIKV exposure at implantation, hatched blastocysts were incubated with 3.26log10, 4.26log10, or 5.26log10 PFU, or not exposed to ZIKV, followed by extended embryo culture for 10 days. ZIKV exposure negatively impacted attachment, growth, and survival in comparison to controls, with exposure to 5.26log10 PFU ZIKV resulting in embryonic degeneration by day 2. Embryonic secretion of pregnancy hormones was lower in ZIKV-exposed embryos. Increasing levels of infectious virus were detected in the culture media post-exposure, suggesting that the trophectoderm is susceptible to productive ZIKV infection. These results demonstrate that ZIKV exposure severely impacts the zona-free blastocyst, whereas exposure at the time of fertilization does not hinder blastocyst formation. Overall, early stages of pregnancy may be profoundly sensitive to infection and pregnancy loss, and the negative impact of ZIKV infection on pregnancy outcomes may be underestimated., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Leucine-rich repeat kinase 2 (LRRK2) G2019S is a relatively common mutation, associated with 1-3% of Parkinson's disease (PD) cases worldwide. G2019S is hypothesized to increase LRRK2 kinase activity. Dopaminergic neurons derived from induced pluripotent stem cells of PD patients carrying LRRK2 G2019S are reported to have several phenotypes compared to wild type controls, including increased activated caspase-3 and reactive oxygen species (ROS), autophagy dysfunction, and simplification of neurites. The common marmoset is envisioned as a candidate nonhuman primate species for comprehensive modeling of genetic mutations. Here, we report our successful use of CRISPR/Cas9 with repair template-mediated homology directed repair to introduce the LRRK2 G2019S mutation, as well as a truncation of the LRRK2 kinase domain, into marmoset embryonic and induced pluripotent stem cells. We found that, similar to humans, marmoset LRRK2 G2019S resulted in elevated kinase activity. Phenotypic evaluation after dopaminergic differentiation demonstrated LRRK2 G2019S-mediated increased intracellular ROS, decreased neuronal viability, and reduced neurite complexity. Importantly, these phenotypes were not observed in clones with LRRK2 truncation. These results demonstrate the feasibility of inducing monogenic mutations in common marmosets and support the use of this species for generating a novel genetic-based model of PD that expresses physiological levels of LRRK2 G2019S.

Ferumoxytol is a superparamagnetic iron oxide nanoparticle used off-label as an intravascular magnetic resonance imaging (MRI) contrast agent. Additionally, ferumoxytol-uptake by macrophages facilitates detection of inflammatory sites by MRI through ferumoxytol-induced image contrast changes. Therefore, ferumoxytol-enhanced MRI holds great potential for assessing vascular function and inflammatory response, critical to determine placental health in pregnancy. This study sought to assess the fetoplacental unit and selected maternal tissues, pregnancy outcomes, and fetal well-being after ferumoxytol administration. In initial developmental studies, seven pregnant rhesus macaques were imaged with or without ferumoxytol administration. Pregnancies went to term with vaginal delivery and infants showed normal growth rates compared to control animals born the same year that did not undergo MRI. To determine the impact of ferumoxytol on the maternal-fetal interface (MFI), fetal well-being, and pregnancy outcome, four pregnant rhesus macaques at ~100 gestational day underwent MRI before and after ferumoxytol administration. Collection of the fetoplacental unit and selected maternal tissues was performed 2-3 days following ferumoxytol administration. A control group that did not receive ferumoxytol or MRI was used for comparison. Iron levels in fetal and MFI tissues did not differ between groups, and there was no significant difference in tissue histopathology with or without exposure to ferumoxytol, and no effect on placental hormone secretion. Together, these results suggest that the use of ferumoxytol and MRI in pregnant rhesus macaques does not negatively impact the MFI and can be a valuable experimental tool in research with this important animal model., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: 3D chemical shift-encoded (CSE)-MRI techniques enable assessment of ferumoxytol concentration but are unreliable in the presence of motion., Purpose: To evaluate a motion-robust 2D-sequential CSE-MRI for R2* and B0 mapping in ferumoxytol-enhanced MRI of the placenta., Study Type: Prospective., Animal Model: Pregnant rhesus macaques., Field Strength/sequence: 3.0T/CSE-MRI., Assessment: 2D-sequential CSE-MRI was compared with 3D respiratory-gated CSE-MRI in placental imaging of 11 anesthetized animals at multiple timepoints before and after ferumoxytol administration, and in ferumoxytol phantoms (0 μg/mL-440 μg/mL). Motion artifacts of CSE-MRI in 10 pregnant women without ferumoxytol administration were assessed retrospectively by three blinded readers (4-point Likert scale). The repeatability of CSE-MRI in seven pregnant women was also prospectively studied., Statistical Tests: Placental R2* and boundary B0 field measurements (ΔB0) were compared between 2D-sequential and 3D respiratory-gated CSE-MRI using linear regression and Bland-Altman analysis., Results: In phantoms, a slope of 0.94 (r 2 = 0.99, concordance correlation coefficient ρ = 0.99), and bias of -4.8 s -1 (limit of agreement [LOA], -41.4 s -1 , +31.8 s -1 ) in R2*, and a slope of 1.07 (r 2 = 1.00, ρ = 0.99) and bias of 11.4 Hz (LOA -12.0 Hz, +34.8 Hz) in ΔB0 were obtained in 2D CSE-MRI compared with 3D CSE-MRI for reference R2* ≤390 s -1 . In animals, a slope of 0.92 (r 2 = 0.97, ρ = 0.98) and bias of -2.2 s -1 (LOA -55.6 s -1 , +51.3 s -1 ) in R2*, and a slope of 1.05 (r 2 = 0.95, ρ = 0.97) and bias of 0.4 Hz (LOA -9.0 Hz, +9.7 Hz) in ΔB0 were obtained. In humans, motion-impaired R2* maps in 3D CSE-MRI (Reader 1: 1.8 ± 0.6, Reader 2: 1.3 ± 0.7, Reader 3: 1.9 ± 0.6), while 2D CSE-MRI was motion-free (Reader 1: 2.9 ± 0.3, Reader 2: 3.0 ± 0, Reader 3: 3.0 ± 0). A mean difference of 0.66 s -1 and coefficient of repeatability of 9.48 s -1 for placental R2* were observed in the repeated 2D CSE-MRI., Data Conclusion: 2D-sequential CSE-MRI provides accurate R2* and B0 measurements in ferumoxytol-enhanced placental MRI of animals in the presence of respiratory motion, and motion-robustness in human placental imaging., Level of Evidence: 1 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2020;51:580-592., (© 2019 International Society for Magnetic Resonance in Medicine.)

Objectives: To assess the feasibility of ferumoxytol-enhanced MRI in pregnancy with a nonhuman primate model., Materials and Methods: In this prospective study, eleven pregnant rhesus macaques at day 98 ± 5 of gestation were divided into three groups, untreated control (UC) (n = 3), saline control (SC) (n = 4) and interleukin 1 beta (IL-1β) treated (IT) (n = 4), which were administered with either saline or IL-1β into the amniotic fluid. All animals were imaged at multiple time points before and after ferumoxytol administration (4 mg/kg). Longitudinal R2* and susceptibility of tissues were obtained using region-of-interest analysis and the longitudinal changes were assessed using linear mixed models and Student's t-test., Results: In fetuses, a slope of 0.3 s - 1 /day (P = 0.008), 0.00 ppm/day (P = 0.699) and - 0.2 s - 1 /day (P = 0.023) was observed in liver R2*, liver susceptibility, and lung R2*, respectively. In placentas, R2* and susceptibility increased immediately after ferumoxytol administration (P < 0.001) and decreased to baseline within two days. The mean change from baseline showed no significant difference between the SC group and the IT group at all scan time points. In maternal livers, R2* increased immediately after ferumoxytol administration, further increased at one-day, and then decreased but remained elevated (P < 0.001). The mean change from baseline showed no significant difference between the SC group and the IT group at all scan time points., Conclusions: This work demonstrates the feasibility of quantitative ferumoxytol-enhanced MRI to measure dynamics of ferumoxytol delivery and washout in the placenta. Stable MRI measurements indicated no evidence of iron deposition in fetal tissues of nonhuman primates after maternal ferumoxytol exposure., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Zika virus (ZIKV) and nonhuman primates have been inextricably linked since the virus was first discovered in a sentinel rhesus macaque in Uganda in 1947. Soon after ZIKV was epidemiologically associated with birth defects in Brazil late in 2015, researchers capitalized on the fact that rhesus macaques are commonly used to model viral immunity and pathogenesis, quickly establishing macaque models for ZIKV infection. Within months, the susceptibility of pregnant macaques to experimental ZIKV challenge and ZIKV-associated abnormalities in fetuses was confirmed. This review discusses key unanswered questions in ZIKV immunity and in the pathogenesis of thecongenital Zika virus syndrome. We focus on those questions that can be best addressed in pregnant nonhuman primates and lessons learned from developing macaque models for ZIKV amid an active epidemic.

Listeria monocytogenes (Lm) is a common environmental bacterium that thrives on vegetation and soil matter, but can infect humans if contaminated food products are ingested, resulting in severe disease in immunosuppressed populations, including pregnant women and newborns. To better understand how the unique immunological milieu of pregnancy increases susceptibility to infection, we study listeriosis in cynomolgus macaques, a non-human primate that closely resembles humans in placentation and in the physiology, and immunology of pregnancy. Non-human primates are naturally susceptible to Lm infection, and spontaneous abortions due to listeriosis are known to occur in outdoor macaque colonies, making them ideal models to understand the disease pathogenesis and host-pathogen relationship of listeriosis. We have previously shown that Lm infection in the first trimester has a high rate of miscarriage. This study expands on our previous findings by assessing how the quantity of Lm as well as stage of pregnancy at the time of exposure may influence disease susceptibility. In the current study we inoculated a cohort of macaques with a lower dose of Lm than our previous study and although this did not result in fetal demise, there was evidence of in utero inflammation and fetal distress. Animals that were reinfected with an equivalent or higher dose of the same strain of Lm resulted in approximately half of cases continuing to term and half ending in fetal demise. These cases had inconsistent bacterial colonization of the fetal compartment, suggesting that Lm does not need to directly infect the placenta to cause adverse pregnancy outcomes. Timed surgical collection of tissues following inoculation demonstrated that transmission from mother to fetus can occur as soon as 5 days post-inoculation. Lastly, third trimester inoculation resulted in pregnancy loss in 3 out of 4 macaques, accompanied by characteristic pathology and Lm colonization. Collectively, our studies demonstrate that common laboratory culture tests may not always recover Lm despite known maternal ingestion. Notably, we also find it is possible for maternal infection to resolve in some cases with no discernible adverse outcome; however, such cases had evidence of a sterile intrauterine inflammatory response, with unknown consequences for fetal development., (Copyright © 2019 Wolfe, Kerr, Mejia, Simmons, Czuprynski and Golos.)

Purpose: To investigate the correspondence between arterial spin labeling (ASL) flow-sensitive alternating inversion recovery (FAIR) and ferumoxytol DCE MRI for the assessment of placental intervillous perfusion., Methods: Ten pregnant macaques in late second trimester were imaged at 3 T using a 2D ASL FAIR, with and without outer-volume saturation pulses used to control the bolus width, and a 3D ferumoxytol DCE-MRI acquisition. The ASL tagged/control pairs were averaged, subtracted, and normalized to create perfusion ratio maps. Contrast arrival time and uptake slope were estimated by fitting the DCE data to a sigmoid function. Macaques (N = 4) received interleukin-1β to induce inflammation and disrupt perfusion., Results: The FAIR tag modification with outer-volume saturation reduced the median ASL ratio percentage compared with conventional FAIR (0.64% ± 1.42% versus 0.71% ± 2.00%; P < .05). Extended ferumoxytol arrival times (34 ± 25 seconds) were observed across the placenta. No significant DCE signal change was measured in fetal tissue ( - 0.6% ± 3.0%; P = .52) or amniotic fluid (1.9% ± 8.8%; P = .59). High ASL ratio was significantly correlated with early arrival time and high uptake slope (P < .05), but ASL signal was not above noise in late-DCE-enhancing regions. No significant differences were observed in perfusion measurements between the interleukin-1β and controls (P > .05)., Conclusion: The ASL-FAIR and ferumoxytol DCE-MRI methods are feasible to detect early blood delivery to the macaque placenta. Outer volume saturation reduced the high macrovascular ASL signal. Interleukin-1β exposure did not alter placental intervillous perfusion. An endogenous-labeling perfusion technique is limited due to extended transit times for flow within the placenta beyond the immediate vicinity of the maternal spiral arteries., (© 2018 International Society for Magnetic Resonance in Medicine.)

Background: Pregnancy complications are often associated with poor uteroplacental vascular adaptation and standard diagnostics are unable to reliably quantify flow in all uteroplacental vessels and have poor sensitivity early in gestation., Purpose: To investigate the feasibility of using 4D flow MRI to assess total uteroplacental blood flow in pregnant rhesus macaques as a precursor to human studies., Study Type: Retrospective feasibility study., Animal Model: Fifteen healthy, pregnant rhesus macaques ranging from the 1 st trimester to 3 rd trimester of gestation., Field Strength/sequence: Abdominal 4D flow MRI was performed on a 3.0T scanner with a radially undersampled phase contrast (PC) sequence. Reference ferumoxytol-enhanced angiograms were acquired with a 3D ultrashort echo time sequence with a center-out radial trajectory., Assessment: Repeatability of flow measurements was assessed with scans performed same-day and on consecutive days in the uterine arteries and ovarian veins. In-flow was compared against out-flow in the uterus, umbilical cord, and fetal heart with a conservation of mass analysis. Conspicuity of uteroplacental vessels was qualitatively compared between PC angiograms derived from 4D flow data and ferumoxytol-enhanced angiograms., Statistical Tests: Bland-Altman analysis was used to quantify same-day and consecutive-day repeatability., Results: Same-day flow measurements showed an average difference between scans of 13% in both the uterine arteries and ovarian veins, while consecutive-day measurements showed average differences of 22% and 24%, respectively. Comparisons of in-flow and out-flow showed average differences of 15% in the uterus, 8% in fetal heart, and 15% in the umbilical cord. PC angiograms showed similar depiction of main uteroplacental vessels as high-resolution, ferumoxytol-enhanced angiograms., Data Conclusion: 4D flow MRI could be used in the rhesus macaque for repeatable flow measurements in the uteroplacental and fetal vasculature, setting the stage for future human studies., Level of Evidence: 2 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2019;49:534-545., (© 2018 International Society for Magnetic Resonance in Medicine.)

The biology of fertility, early development, and pregnancy is variable across mammalian species. In addition, while the physiology and pathophysiology of human diseases can be investigated in other animal models (principally, rodents), differences between human and lower mammals often present limitations in the applicability of physiological processes from rodent models to human biology. Since 1984, when the first live birth from rhesus monkey in vitro fertilization and embryo transfer was reported (Bavister et al., Proc Natl Acad Sci 81:2218-2222, 1984), there has been progress in the implementation of assisted reproductive technologies with several nonhuman primate (NHP) species that play important roles in biomedical research. In recent years, the significance of this progress has been amplified by the development of genomic editing approaches for facile genetic manipulation of the embryo, including methods now applied to NHPs (Liu et al., Cell Stem Cell 14:323-328, 2014; Niu et al., Cell 156:836-843, 2014). In this review, we summarize current protocols and practices for the common marmoset. It is our intention to provide current state-of-the-art protocols for gamete procurement and in vitro fertilization techniques, so that laboratories wishing to implement experimental embryology in marmoset models will have a basic set of tools with which to initiate such studies.

Hofbauer cells are placental villous macrophages of fetal origin that are present throughout pregnancy. Although Hofbauer cell populations are antigenically and morphologically heterogeneous, their epigenetic, antigenic, and functional profiles most closely resemble alternatively activated macrophages or what are referred to as M2a, M2b, M2c, and M2d polarity subtypes. Consistent with an M2-like profile, these cells play an important role in placental development including vasculogenesis and angiogenesis. During placental inflammation Hofbauer cells may produce pro-inflammatory cytokines or mediators that damage the villous cell barrier, and induce fibrotic responses within the villi as a continuum of chronic inflammation. However, to date, there is no evidence that Hofbauer cells become classically activated or adopt an M1 polarity phenotype that is able to kill microbes. To the contrary, their predominant M2 like qualities may be why these cells are ineffective in controlling most TORCH infections. Moreover, Hofbauer cells may contribute to vertical transmission of various pathogens to the fetus since they can harbor live virus and serve as reservoirs within the placenta. The goal of this review is to summarize what is currently known about the role of Hofbauer cells in normal and complicated pregnancies that involve immunologic disorders, inflammation, and/or infection.