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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.

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.

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.

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.

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., Author summary Zika virus (ZIKV) gained global attention during an explosive outbreak in the Americas in 2015–16 when it was causally associated with the constellation of birth defects now termed congenital Zika syndrome (CZS). However, a substantial proportion of gestational ZIKV infections result in babies without apparent birth defects. Could there be other factors that influence ZIKV pathogenicity? For example, it is well-established that pre-existing immunity to one dengue virus (DENV) serotype can enhance the severity of a secondary DENV infection. ZIKV is antigenically closely related to DENV, but whether DENV-specific antibodies enhance the severity of ZIKV infection is unclear. To answer this question, we used our non-human primate model of ZIKV to assess the impact of pre-existing immunity to DENV on ZIKV pathogenesis during pregnancy. We did not observe any difference in ZIKV replication in plasma between macaques that were immune to DENV and those that were not. However, there was more ZIKV vRNA detected in the placenta of macaques immune to DENV, suggesting DENV immunity could enhance ZIKV infection of the placenta. As vaccines to both DENV and ZIKV are developed, it remains critical to understand the risks of DENV immunity for pregnant women exposed to ZIKV.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2018 there were nine themed workshops, five of which are summarised in this report. These workshops discussed new perspectives and knowledge in the following areas of research: 1) preeclampsia; 2) abnormally invasive placenta; 3) placental infection; 4) gestational trophoblastic disease; 4) drug delivery to treat placental dysfunction.

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 (r2 = 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 (r2 = 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 (r2 = 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 (r2 = 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.

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.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a potentially curative treatment for hematologic cancers and chronic infections such as human immunodeficiency virus (HIV). Its success in these settings is attributed to the ability of engrafting immune cells to eliminate cancer cells or deplete the HIV reservoir (graft-versus-host effect [GvHE]). However, alloHSCT is commonly associated with graft-versus-host diseases (GvHDs) causing significant morbidity and mortality, thereby requiring development of novel allogeneic HSCT protocols and therapies promoting GvHE without GvHD using physiologically relevant preclinical models. Here we evaluated the outcomes of major histocompatibility complex-matched T-cell receptor α/β-depleted alloHSCT in Mauritian cynomolgus macaques (MCMs). Following T-cell receptor α/β depletion, bone marrow cells were transplanted into major histocompatibility complex-identical MCMs conditioned with total body irradiation. GvHD prophylaxis included sirolimus alone in two animals or tacrolimus with cyclophosphamide in another two animals. Posttransplant chimerism was determined by sequencing diagnostic single-nucleotide polymorphisms to quantify the amounts of donor and recipient cells present in blood. Animals treated posttransplant with sirolimus developed nearly complete chimerism with acute GvHD. In the cyclophosphamide and tacrolimus treatment group, animals developed mixed chimerism without GvHD, with long-term engraftment observed in one animal. None of the animals developed cytomegalovirus infection. These studies indicate the feasibility of alloHSCT engraftment without GvHD in an MHC-identical MCM model following complete myeloablative conditioning and anti-GvHD prophylaxis with posttransplant cyclophosphamide and tacrolimus. Further exploration of this model will provide a platform for elucidating the mechanisms of GvHD and GvHE and for testing novel alloHSCT modalities for HIV infection.

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.

Zika virus (ZIKV) infection in humans has been associated with severe congenital defects (i.e. microcephaly) and pregnancy loss. Here we show that 26% of nonhuman primates infected with Asian/American ZIKV in early gestation experienced fetal demise later in pregnancy despite few clinical signs of infection. Pregnancy loss due to asymptomatic ZIKV infection may therefore be a common but under-recognized adverse outcome related to maternal ZIKV infection.

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_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.

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.Summary SentenceFerumoxytol magnetic resonance imaging for non-invasive pregnancy monitoring of the rhesus macaque does not impact histopathology or iron content of the maternal–fetal interface.

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 0.3s(−1)/day (P = 0.008), 0.00ppm/day (P = 0.699) and −0.2s(−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.

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.

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.

Problem Immune cells within the endometrium at implantation are thought to play an important role in implantation, although their exact role is not well understood. Method of study A co-culture system of rhesus monkey embryos and maternal immune cells was established. Blastocysts obtained by in vitro fertilization were co-cultured with peripheral blood cells or decidual macrophages. Culture media were collected to assess secretions. Embryo growth was monitored, and trophoblasts were evaluated for proliferation, apoptosis, and differentiation. Results Embryonic trophoblast outgrowths were visible within 6 days of culture, and the area of embryo outgrowth was reduced when blastocysts were cultured with peripheral-derived or decidual macrophages. Trophoblast proliferation was not significantly affected with macrophage co-culture while chorionic gonadotropin secretion was increased. Trophoblast expression of CDH 11 and GJA1 was increased, suggesting that macrophages accelerate differentiation of peri-implantation trophoblasts. Conclusions These results indicate an important role of macrophages in placentation and pregnancy success.

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.

PURPOSE: To investigate the correspondence between arterial spin labeling (ASL) flow-sensitive alternating inversion recovery (FAIR) and ferumoxytol dynamic contrast enhanced (DCE) magnetic resonance imaging (MRI) for the assessment of placental intervillous perfusion. METHODS: Ten pregnant macaques in late 2(nd) trimester were imaged at 3T using a 2D ASL FAIR, with and without outer volume saturation (OVS) pulses used to control the bolus width, and a 3D ferumoxytol DCE MRI acquisition. ASL tagged/control pairs were averaged, subtracted, and normalized to create perfusion ratio maps. Contrast arrival time and uptake slope were estimated by fitting DCE data to a sigmoid function. Macaques (N=4) received interleukin-1β to induce inflammation and disrupt perfusion. RESULTS: FAIR tag modification with OVS reduced median ASL ratio percentage compared to conventional FAIR (0.64±1.42% vs. 0.71±2.00%;p0.05). CONCLUSION: ASL FAIR and ferumoxytol DCE MRI are feasible methods to detect early blood delivery to the macaque placenta. OVS reduced 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.

The rhesus macaque is an important animal model for AIDS and other infectious diseases; however, studies to address NK cell function in this species have been limited by the lack of defined ligands for killer cell Ig-like receptors (KIRs). To identify ligands for rhesus macaque KIRs, we adopted a novel approach based on a pair of stable cell lines. NFAT-responsive luciferase reporter cell lines expressing the extracellular domains of macaque KIRs fused to the transmembrane and cytoplasmic domains of CD28 and CD3ζ were incubated with target cells expressing individual MHC class I molecules, and ligand recognition was detected by the MHC class I–dependent upregulation of luciferase. Using this approach, we found that Mamu-KIR3DL01, -KIR3DL06, -KIR3DL08, and -KIR3DSw08 all recognize Mamu-Bw4 molecules but with differing allotype specificity. In contrast, Mamu-KIR3DL05 recognizes Mamu-A and Mamu-A–related molecules, including Mamu-A1*002 and -A3*13, Mamu-B*036, the product of a recombinant Mamu-B allele with α1 and α2 domain sequences derived from a MHC-A gene, and Mamu-AG*01, a nonclassical molecule expressed on placental trophoblasts that originated from an ancestral duplication of a MHC-A gene. These results reveal an expansion of the lineage II KIRs in macaques that recognize Bw4 ligands and identify a nonclassical molecule implicated in placental development and pregnancy as a ligand for Mamu-KIR3DL05. In addition to offering new insights into KIR–MHC class I coevolution, these findings provide an important foundation for investigating the role of NK cells in the rhesus macaque as an animal model for infectious diseases and reproductive biology.

Background The initiation of primate embryo invasion into the endometrium and the formation of the placenta from trophoblasts, fetal mesenchyme, and vascular components are essential for the establishment of a successful pregnancy. The mechanisms which direct morphogenesis of the chorionic villi, and the interactions between trophectoderm-derived trophoblasts and the fetal mesenchyme to direct these processes during placentation are not well understood due to a dearth of systems to examine and manipulate real-time primate implantation. Here we describe an in vitro three-dimensional (3-D) model to study implantation which utilized IVF-generated rhesus monkey embryos cultured in a Matrigel explant system. Methods Blastocyst stage embryos were embedded in a 3-D microenvironment of a Matrigel carrier and co-cultured with a feeder layer of cells generating conditioned medium. Throughout the course of embryo co-culture embryo growth and secretions were monitored. Embedded embryos were then sectioned and stained for markers of trophoblast function and differentiation. Results Signs of implantation were observed including enlargement of the embryo mass, and invasion and proliferation of trophoblast outgrowths. Expression of chorionic gonadotropin defined by immunohistochemical staining, and secretion of chorionic gonadotropin and progesterone coincident with the appearance of trophoblast outgrowths, supported the conclusion that a trophoblast cell lineage formed from implanted embryos. Positive staining for selected markers including Ki67, MHC class I, NeuN, CD31, vonWillebrand Factor and Vimentin, suggest growth and differentiation of the embryo following embedding. Conclusions This 3-D in vitro system will facilitate further study of primate embryo biology, with potential to provide a platform for study of genes related to implantation defects and trophoblast differentiation. Electronic supplementary material The online version of this article (10.1186/s12958-018-0340-3) contains supplementary material, which is available to authorized users.

Congenital Zika virus (ZIKV) infection impacts fetal development and pregnancy outcomes. We infected a pregnant rhesus macaque with a Puerto Rican ZIKV isolate in the first trimester. The pregnancy was complicated by preterm premature rupture of membranes (PPROM), intraamniotic bacterial infection and fetal demise 49 days post infection (gestational day 95). Significant pathology at the maternal-fetal interface included acute chorioamnionitis, placental infarcts, and leukocytoclastic vasculitis of the myometrial radial arteries. ZIKV RNA was disseminated throughout fetal tissues and maternal immune system tissues at necropsy, as assessed by quantitative RT-PCR for viral RNA. Replicating ZIKV was identified in fetal tissues, maternal uterus, and maternal spleen by fluorescent in situ hybridization for viral replication intermediates. Fetal ocular pathology included a choroidal coloboma, suspected anterior segment dysgenesis, and a dysplastic retina. This is the first report of ocular pathology and prolonged viral replication in both maternal and fetal tissues following congenital ZIKV infection in a rhesus macaque. PPROM followed by fetal demise and severe pathology of the visual system have not been described in macaque congenital ZIKV infection previously. While this case of ZIKV infection during pregnancy was complicated by bacterial infection with PPROM, the role of ZIKV on this outcome cannot be precisely defined, and further nonhuman primate studies will determine if increased risk for PPROM or other adverse pregnancy outcomes are associated with congenital ZIKV infection.

Objective Expression of microRNAs (miRNAs) in the human placenta is dynamic across gestation, with expression of miRNAs belonging to the C14MC, C19MC and miR-371-3 clusters. Specifically, miRNAs within the C19MC cluster are exclusively expressed in primates with predominant expression in the placenta. Non-human primates can be utilized to study developmental processes of placentation in vivo that cannot be assessed in the human placenta, however, miRNA expression has not been defined in the macaque placenta. Our objective was to profile miRNAs in the macaque placenta, hypothesizing that expression is conserved between the macaque and human placenta. Methods Total RNA from first trimester and term macaque placentas (n = 4 per group) was analyzed through RNA-sequencing and validated by quantitative real-time PCR (qRT-PCR). Results A total of 607 pre-miRNAs previously annotated in the macaque reference database (miRBase21) were detected, and 166 miRNAs were differentially expressed between first trimester and term placentas. A total of 457 unannotated sequences were detected and deemed candidate novel miRNAs by miRDeep2 software. Differential expression was confirmed for six of nine miRNAs evaluated by qRT-PCR. Comparative analysis demonstrated expression of several miRNA orthologs of human pregnancy-associated miRNA clusters in the macaque placenta. Conclusions Profiling placental miRNAs of the macaque revealed conserved expression of a number of miRNAs within the C14MC, C19MC and miR-371-3 clusters between the human and macaque. These results establish non-human primates as a model for human placentation and miRNA biology, with the prediction of their functional significance in placental development and function.

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 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.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2016 there were twelve themed workshops, four of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of decidual-trophoblast interaction, regulation of trophoblast invasion, immune cells at the maternal-fetal interface, and placental inflammation.

Infection with Zika virus (ZIKV) is associated with human congenital fetal anomalies. To model fetal outcomes in nonhuman primates, we administered Asian-lineage ZIKV subcutaneously to four pregnant rhesus macaques. While non-pregnant animals in a previous study contemporary with the current report clear viremia within 10-12 days, maternal viremia was prolonged in 3 of 4 pregnancies. Fetal head growth velocity in the last month of gestation determined by ultrasound assessment of head circumference was decreased in comparison with biparietal diameter and femur length within each fetus, both within normal range. ZIKV RNA was detected in tissues from all four fetuses at term cesarean section. In all pregnancies, neutrophilic infiltration was present at the maternal-fetal interface (decidua, placenta, fetal membranes), in various fetal tissues, and in fetal retina, choroid, and optic nerve (first trimester infection only). Consistent vertical transmission in this primate model may provide a platform to assess risk factors and test therapeutic interventions for interruption of fetal infection. The results may also suggest that maternal-fetal ZIKV transmission in human pregnancy may be more frequent than currently appreciated.Author summaryMaternal ZIKV infection in pregnancy is associated with severe fetal anomalies, including microcephaly. It has been shown that infection manifests differently in pregnancy than in the non-pregnant state, with prolonged maternal viremia. ZIKV is spread by mosquitos and through sexual contact and since its first detection in early 2015, has become endemic to the Americas. While much has been learned from studying infected human pregnancies, there are still many questions concerning transmission of ZIKV from mother to fetus. Investigating ZIKV infection in non-human primates could help answer these questions due to similarities in the immune system, and the tissues separating the fetus from the mother during pregnancy. Our study serves to model ZIKV transmission in early and late pregnancy, as well as study the effects of this infection on the fetus and mother at these different times in pregnancy. The data collected provides an important insight on ZIKV in pregnancy where the pregnancies have been monitored throughout the entire infection period until term, and suggests that vertical transmission may be very efficient, although severe fetal outcomes are uncommon.

Infection with Listeria monocytogenes during pregnancy is associated with miscarriage, preterm birth, and neonatal complications, including sepsis and meningitis. While the risk of these conditions is thought to be greatest during the third trimester of pregnancy, the determinants of fetoplacental susceptibility to infection, the contribution of gestational age, and the in vivo progression of disease at the maternal-fetal interface are poorly understood. We developed a nonhuman primate model of listeriosis to better understand antecedents of adverse pregnancy outcomes in early pregnancy. Four pregnant cynomolgus macaques (Macaca fascicularis) received a single intragastric inoculation between days 36 and 46 of gestation with 107 CFU of an L. monocytogenes strain isolated from a previous cluster of human listeriosis cases that resulted in adverse pregnancy outcomes. Fecal shedding, maternal bacteremia, and fetal demise were consistently noted within 7 to 13 days. Biopsy specimens of maternal liver, spleen, and lymph node displayed variable inflammation and relatively low bacterial burden. In comparison, we observed greater bacterial burden in the decidua and placenta and the highest burden in fetal tissues. Histopathology indicated vasculitis, fibrinoid necrosis, and thrombosis of the decidual spiral arteries, acute chorioamnionitis and villitis in the placenta, and hematogenous infection of the fetus. Vascular pathology suggests early impact of L. monocytogenes infection on spiral arteries in the decidua, which we hypothesize precipitates subsequent placentitis and fetal demise. These results demonstrate that L. monocytogenes tropism for the maternal reproductive tract results in infection of the decidua, placenta, and the fetus itself during the first trimester of pregnancy., IMPORTANCE Although listeriosis is known to cause significant fetal morbidity and mortality, it is typically recognized in the third trimester of human pregnancy. Its impact on early pregnancy is poorly defined. Here we provide evidence that exposure to L. monocytogenes in the first trimester poses a greater risk of fetal loss than currently appreciated. Similarities in human and nonhuman primate placentation, physiology, and reproductive immunology make this work highly relevant to human pregnancy. We highlight the concept that the maternal immune response that protects the mother from serious disease is unable to protect the fetus, a concept relevant to classic TORCH (toxoplasmosis, other, rubella, cytomegalovirus, and herpes) infections and newly illuminated by current Zika virus outbreaks. Studies with this model, using the well-understood organism L. monocytogenes, will permit precise analysis of host-pathogen interactions at the maternal-fetal interface and have broad significance to both recognized and emerging infections in the setting of pregnancy.

The primitive cardiac tube starts beating 6-8 weeks post fertilization in the developing embryo. In order to describe normal cardiac development during late first and early second trimester in human fetuses this study used microarray and pathways analysis and created a corresponding 'normal' database. Fourteen fetal hearts from human fetuses between 10 and 18 weeks of gestational age (GA) were prospectively collected at the time of elective termination of pregnancy. RNA from recovered tissues was used for transcriptome analysis with Affymetrix 1.0 ST microarray chip. From the amassed data we investigated differences in cardiac development within the 10-18 GA period dividing the sample by GA in three groups: 10-12 (H1), 13-15 (H2) and 16-18 (H3) weeks. A fold change of 2 or above adjusted for a false discovery rate of 5% was used as initial cutoff to determine differential gene expression for individual genes. Test for enrichment to identify functional groups was carried out using the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Array analysis correctly identified the cardiac specific genes, and transcripts reported to be differentially expressed were confirmed by qRT-PCR. Single transcript and Ontology analysis showed first trimester heart expression of myosin-related genes to be up-regulated >5-fold compared with second trimester heart. In contrast the second trimester hearts showed further gestation-related increases in many genes involved in energy production and cardiac remodeling. In conclusion, fetal heart development during the first trimester was dominated by heart-specific genes coding for myocardial development and differentiation. During the second trimester, transcripts related to energy generation and cardiomyocyte communication for contractile coordination/proliferation were more dominant. Transcripts related to fatty acid metabolism can be seen as early as 10 weeks and clearly increase as the heart matures. Retinol receptor and gamma-aminobutyric acid (GABA) receptor transcripts were detected, and have not been described previously in human fetal heart during this period. For the first time global gene expression of heart has been described in human samples to create a database of normal development to understand and compare with known abnormal fetal heart development.

The development of the placenta is imperative for successful pregnancy establishment, yet the earliest differentiation events of the blastocyst-derived trophectoderm that forms the placenta remain difficult to study in humans. Human embryonic stem cells (hESC) display a unique ability to form trophoblast cells when induced to differentiate either by the addition of exogenous BMP4 or by the formation of cellular aggregates called embryoid bodies. While mouse trophoblast stem cells (TSC) have been isolated from blastocyst outgrowths, mouse ESC do not spontaneously differentiate into trophoblast cells. In this review, we focus on addressing the similarities and differences between mouse TSC differentiation and hESC-derived trophoblast differentiation. We discuss the functional and mechanistic diversity that is found in different species models. Of central importance are the unique signaling events that trigger downstream gene expression that create specific cellular fate decisions. We support the idea that we must understand the nuances that hESC differentiation models display so that investigators can choose the appropriate model system to fit experimental needs.

Background Zika virus (ZIKV; Flaviviridae, Flavivirus) was declared a public health emergency of international concern by the World Health Organization (WHO) in February 2016, because of the evidence linking infection with ZIKV to neurological complications, such as Guillain-Barre Syndrome in adults and congenital birth defects including microcephaly in the developing fetus. Because development of a ZIKV vaccine is a top research priority and because the genetic and antigenic variability of many RNA viruses limits the effectiveness of vaccines, assessing whether immunity elicited against one ZIKV strain is sufficient to confer broad protection against all ZIKV strains is critical. Recently, in vitro studies demonstrated that ZIKV likely circulates as a single serotype. Here, we demonstrate that immunity elicited by African lineage ZIKV protects rhesus macaques against subsequent infection with Asian lineage ZIKV. Methodology/Principal Findings Using our recently developed rhesus macaque model of ZIKV infection, we report that the prototypical ZIKV strain MR766 productively infects macaques, and that immunity elicited by MR766 protects macaques against heterologous Asian ZIKV. Furthermore, using next generation deep sequencing, we found in vivo restoration of a putative N-linked glycosylation site upon replication in macaques that is absent in numerous MR766 strains that are widely being used by the research community. This reversion highlights the importance of carefully examining the sequence composition of all viral stocks as well as understanding how passage history may alter a virus from its original form. Conclusions/Significance An effective ZIKV vaccine is needed to prevent infection-associated fetal abnormalities. Macaques whose immune responses were primed by infection with East African ZIKV were completely protected from detectable viremia when subsequently rechallenged with heterologous Asian ZIKV. Therefore, these data suggest that immunogen selection is unlikely to adversely affect the breadth of vaccine protection, i.e., any Asian ZIKV immunogen that protects against homologous challenge will likely confer protection against all other Asian ZIKV strains., Author Summary Zika virus (ZIKV) isolates are genetically diverse, but belong to two recognized lineages, termed “African” and “Asian.” Asian ZIKV infection during pregnancy causes fetal abnormalities including microcephaly. Developing an effective preventative Zika virus vaccine that protects pregnant women is essential for minimizing fetal abnormalities; at least 18 groups are developing ZIKV vaccines [1]. The genetic and antigenic variability of many RNA viruses limits the effectiveness of vaccines, and the degree to which immunity against one ZIKV strain could provide protection against another is unknown. Here we show that rhesus macaques infected with the East African ZIKV strain MR766 are completely protected from subsequent infection with heterologous Asian ZIKV. MR766 is more genetically divergent from all known Asian ZIKV strains than Asian ZIKV strains are from one another. Therefore, ZIKV strain selection is unlikely to compromise vaccine effectiveness.

Infection with Asian-lineage Zika virus (ZIKV) has been associated with Guillain–Barré syndrome and fetal abnormalities, but the underlying mechanisms remain poorly understood. Animal models of infection are thus urgently needed. Here we show that rhesus macaques are susceptible to infection by an Asian-lineage ZIKV closely related to strains currently circulating in the Americas. Following subcutaneous inoculation, ZIKV RNA is detected in plasma 1 day post infection (d.p.i.) in all animals (N=8, including 2 pregnant animals), and is also present in saliva, urine and cerebrospinal fluid. Non-pregnant and pregnant animals remain viremic for 21 days and for up to at least 57 days, respectively. Neutralizing antibodies are detected by 21 d.p.i. Rechallenge 10 weeks after the initial challenge results in no detectable virus replication, indicating protective immunity against homologous strains. Therefore, Asian-lineage ZIKV infection of rhesus macaques provides a relevant animal model for studying pathogenesis and evaluating potential interventions against human infection, including during pregnancy., Animal models of infection with Zika virus (ZIKV) are urgently needed for a better understanding of pathogenesis and for testing potential therapies. Here, the authors describe infection of rhesus macaques with an Asian-lineage ZIKV strain as a relevant animal model for studying ZIKV pathogenesis.

Problem Throughout the reproductive cycle and into early pregnancy, the normal endometrium undergoes changes in a range of leukocytes, epithelia, stromal fibroblasts, and vascular structures caused by intersecting effects of hormone balance and embryo implantation. The direct investigation in humans of reproductive tract responses during normal and physiologically altered cycles is not practical or feasible. Method and Study The aim of this study was to define immunological and morphological changes through immunohistological and morphometric evaluation of the endometrium throughout the menstrual cycle and the decidua during early gestation in the rhesus monkey, a tractable experimental animal model. Results A zone-dependent method for the immunohistological description of the rhesus uterine mucosa was established and showed that leukocyte infiltration, stromal cell decidualization, glandular and vascular responses were zone- and cell type-dependent, and changed throughout the cycle and early pregnancy. Morphological heterogeneity of uterine natural killer cells in the cycling endometrium and gestational decidua were consistent with the recent characterization of phenotypic subsets. Conclusions These data establish a morphological platform upon which to further study the regulation of endometrial responses to the hormonal mileau of pregnancy, the control of local leukocyte populations, and the responses to threatened pregnancy, infection, and inflammation.

Studies of early placental development in humans are difficult because of limitations on experimental material availability from the perimplantation period. We used a coculture system to determine the effects of various effector cell types on trophoblast differentiation. Enhanced green fluorescent protein (EGFP)-expressing H1 human embryonic stem cells were used in co-suspension with human term placental fibroblasts (TPFs) and dermal fibroblasts (CI2F) to form combination embryoid bodies (EBs), with the goal of recapitulating placental morphogenesis through incorporation of placental mesenchymal cells. Overall, the results demonstrated that when using mesenchymal cells for EB preparation from term placentas (TPF), combination EB-derived trophoblasts secrete higher levels of human chorionic gonadotropin (hCG) and progesterone compared to EBs made without effector cells, whereas there was no effect of dermal fibroblasts. This is due to the secretory activity of the EB-derived trophoblasts and not due to the number of differentiated trophoblasts per EB, demonstrating that nontrophoblast cells of the placenta can influence trophoblast endocrine activity.

While the determination of the trophoblast lineage and the facilitation of placental morphogenesis by trophoblast interactions with other cells of the placenta are crucial components for the establishment of pregnancy, these processes are not tractable at the time of human implantation. Embryonic stem cells (ESCs) provide an embryonic surrogate to derive insights into these processes. In this review, we will summarize current paradigms which promote trophoblast differentiation from ESCs, and potential opportunities for their use to further define signals directing morphogenesis of the placenta following implantation of the embryo into the endometrium.

While there is broad agreement that interactions of the human maternal immune system with the tissues and cells of the implanting embryo are likely to be critical contributors to pregnancy success, there remains a dearth of information which directly confirms this expectation. Although animal models of reproductive function often provide opportunities for confirming such hypotheses, progress in this area has been sporadic due to limitations of traditional laboratory or agricultural animal models, such as rodents, sheep, pigs and cattle. Many of these limitations derive from divergent modes of implantation and placentation across mammalian species. Over the past decade there has been progress in the development of the nonhuman primate as a model in which to address questions of pregnancy success in the area of immunology. The purpose of this review is to compare available model species, summarize current knowledge and recent progress with an emphasis on experimental in vivo manipulations, and suggest areas available for additional study and growth.

Nonhuman primates are important animal models for the study of the maternal immune response to implantation within the decidua. The objective of this study was to define the placental expression of major histocompatibility complex (MHC) class I molecules in the cynomolgus (Macaca fascicularis) and vervet (African green) (Chlorocebus aethiops) monkeys. Early pregnancy (d36-42) cynomolgus and vervet placentas were obtained by fetectomy and prepared for histological evaluation. A pan-MHC class I monoclonal antibody demonstrated MHC class I expression in both vervet and cynomolgus placental trophoblasts, with particularly high expression in the villous syncytium, as previously shown in the rhesus and baboon. Placental cytotrophoblasts were isolated by enzymatic dispersion and gradient centrifugation and cultured, and multicolor flow cytometry was used to phenotype cell populations. Culture of isolated villous cytotrophoblasts demonstrated that MHC class I expression was linked to syncytiotrophoblast differentiation. A monoclonal antibody against Mamu-AG, the nonclassical MHC class I homolog of HLA-G in the rhesus monkey, demonstrated intense immunostaining and cell surface expression in cynomolgus placental trophoblasts; however, staining with vervet placenta and cells was low and inconsistent. Reverse transcriptase polymerase chain reaction was used to clone MHC class I molecules expressed in cynomolgus and vervet placentas. While Mafa-AG messenger RNA (mRNA) was readily detectable in cynomolgus placental RNA and was99% identical at the amino acid level with Mamu-AG, 7/8 Chae-AG complementary DNAs had an unusual 16 amino acid repeat in the alpha1 domain, and all clones had an unexpected absence of the early stop codon at the 3'-end of the mRNA diagnostic for rhesus, cynomolgus, and baboon AG mRNAs, as well as HLA-G. We conclude that while the vervet monkey has retained the placental expression of a primate-specific nonclassical MHC class I locus, diversity is also revealed in this locus expressed at the maternal-fetal interface, thought to participate in placental regulation of the maternal immune response to embryo implantation and pregnancy.

Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial and rate-limiting step of tryptophan degradation along the kynurenine pathway, and is hypothesized to limit tryptophan availability at embryo implantation and prevent maternal T cell activation at the maternal-fetal interface. To determine if nonhuman primates are suitable models for investigating the role of IDO during pregnancy, we defined the expression of IDO in the rhesus monkey and common marmoset with particular attention to the female reproductive tract and placenta. IDO mRNA was detected by RT-PCR in the rhesus monkey term placenta, lung, small intestine, spleen, lymph node and nonpregnant uterus, and also in the common marmoset placenta. Immunohistochemical analysis of rhesus monkey tissues localized IDO to glandular epithelium of nonpregnant endometrium and first trimester decidua, vessel endothelium of nonpregnant myometrium, first trimester decidua and term decidua, and villous vessel endothelium and syncytiotrophoblast of term placenta. Western blot analysis confirmed IDO in rhesus monkey term placenta. In the common marmoset, IDO was detected in glandular epithelium of the nonpregnant uterus and in the decidua at day 60 and day 128 of gestation. IDO activity was higher in rhesus monkey and common marmoset decidua and placentas than in other tissues. Confirmation of IDO expression in rhesus monkey and common marmoset uterine and placental tissues supports the hypothesis that this enzyme regulates immune activation at the maternal-fetal interface and demonstrates that nonhuman primates may provide models with distinct similarities to human placentation to study the role of IDO in maternal-fetal immune dialogue.

Leaders gathered at the US National Institutes of Health in November 2014 to discuss recent advances and emerging research areas in aspects of maternal-fetal immunity that may affect fetal development and pregnancy success.

Leukocyte Ig-like receptors (LILRs) are a family of receptors that have inhibitory and activating functions and widely expressed by lymphoid and myeloid cells. Here we report the identification of the rhesus monkey LILRs by screening of rhesus spleen and decidua cDNA libraries and RT-PCR cloning. We obtained eight different full-length clones with structural and functional diversity similar to human LILRs, including LILRs with immunoreceptor tyrosine-based inhibitory motifs, LILRs with truncated cytoplasmic tails containing positively charged arginine residues in the transmembrane domain, and putative soluble receptors lacking transmembrane or cytoplasmic domains. Characterization of rhesus LILRs will facilitate use of this non-human primate model for the study of the functional role(s) of LILRs, including immune regulation through interaction with non-classical MHC class I molecules.

We performed immunohistochemical analysis of DC-SIGN expression at the maternal–fetal interface at different stages of pregnancy in the rhesus monkey. Natural killer cells, monocytes and macrophages were observed in the nonpregnant endometrium, particularly in the luteal phase, and were increased in pregnant endometrium. No DC-SIGN + cells were observed in the nonpregnant uterus. We observed decidual DC-SIGN + cells within 1 week of implantation, and they increased in number during the first 5 weeks of gestation. DC-SIGN + cells showed a clear differential distribution in the decidua in the first 2 weeks of pregnancy, being found only adjacent to the implantation site, in marked contrast to the widespread distribution of CD68 + macrophages and CD56 + NK cells throughout the endometrium. DC-SIGN + cells also showed a more dendritic morphology than the general CD68 + cell population, and analysis of serial sections indicated an overlapping but not identical localization of these markers. Mature dendritic cells could not be detected as judged by total absence of immunostaining for CD83, CD86, DEC-205, or CD1a. DC-SIGN + cells were defined as MHC class II + and CD14 + by flow cytometry. We conclude that DC-SIGN expression is an early response by the primate maternal immune system to the implanting embryo. The selectively distributed population of DC-SIGN + decidual leukocytes may represent a morphologically and phenotypically distinct subpopulation of decidual macrophages of early pregnancy that could contribute to the establishment of maternal–fetal immune tolerance.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2012 there were twelve themed workshops, three of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of models and technical issues involved in placenta research: 1) comparative placentation and animal models; 2) advanced techniques in placental histopathology; 3) human pluripotent stem cells as a model for trophoblast differentiation.

The transcriptional regulators aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) modulate the transcription of genes involved in cellular differentiation and proliferation. In this study, we investigated the expression of these transcriptional regulators in the female reproductive tract. AHR and ARNT mRNA transcripts were readily detected by a ribonuclease protection assay in all reproductive tissues examined. The expression of these factors in the endometrium and myometrium did not vary during the menstrual cycle, and was not different in pre- versus post-menopausal women. However, post-menopausal women on continuous hormone replacement therapy had greater expression of AHR but not of ARNT in the endometrium and myometrium when compared with women not taking hormones. Leiomyomas expressed significantly less AHR and ARNT mRNA compared with normal myometrium. The ovaries expressed both AHR and ARNT mRNA, and expression was unaffected by age. Endometriotic ovarian cysts expressed more AHR but not more ARNT mRNA compared with healthy ovarian tissue. However, there were no changes in the expression of AHR or ARNT mRNA in ovarian cancer. In conclusion, the female reproductive tract expresses mRNA for the transcription factors AHR and ARNT, and changes in their expression at select target sites in specific pathological conditions such as endometriosis and uterine leiomyomas suggest a potential role for these factors in the pathogenesis of these conditions.

Transgenic mice have provided invaluable information about gene function and regulation. However, because of marked differences between rodents and primates, some areas of human biology such as early embryonic development, aging, and maternal–fetal interactions would be best studied in a nonhuman primate model. Here, we report that gene transfer into rhesus monkey (Macaca mulatta) preimplantation embryos gives rise to transgenic placentas that express a reporter transgene (eGFP). Blastocysts resulting from culture ofin vitrofertilized ova were transduced with a self-inactivating lentiviral vector and transferred into recipient females. One twin and one singleton pregnancy were produced from a single stimulation cycle, and one live rhesus monkey was born from each pregnancy. Placentas from all conceptuses showed expression of the transgene as detected by reverse transcription–PCR, ribonuclease protection assay, direct epifluorescence, immunohistochemistry, and Western blot analysis. Integration in somatic tissues of the offspring was not detected. A maternal immune response to the xenogeneic placental antigen was shown by the presence of anti-GFP antibodies in peripheral blood of the recipient females by day 99 of gestation (term = 165 days). These results demonstrate that transgene expression during gestation is compatible with successful pregnancy in nonhuman primates and provides an approach that could be broadly applicable to the development of novel models for primate biomedical research.

In this study, we carried out a phenotypic and functional characterization of lymphocytes isolated from the uterine endometrium of the pregnant rhesus monkey. A majority (80%) of these cells were CD56 bright+ , CD3− had typical large granular lymphocyte/uterine natural killer (NK) cell morphology and contained numerous cytoplasmic granules. Flow cytometric evaluation showed that rhesus decidual CD56 bright+ cells shared other phenotypic features of human uterine NK cells, including low levels of CD45RA and CD62L expression. A majority of the rhesus uterine CD56 bright+ cells expressed low levels of CD 16 but were CD2−. In contrast, most rhesus CD16+ peripheral blood cells were CD56−. In addition to the primary population of CD56 bright+ cells, a minor subset of smaller and less granular CD56 intermediate+ decidual lymphocytes was identified, the majority of which were CD16−, CD2 + . Decidual CD56+ cells did not express monocyte/macrophage markers, including CD14, CD64 and CD68. Decidual lymphocytes effectively lysed K562, Raji and particularly 721.221 targets in cytotoxicity assays. Together, these results suggest that as in human pregnancy, rhesus decidual CD56 bright+ cells represent a distinct lymphocyte subset that belongs to the NK cell lineage.

Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. Here we report the derivation of a cloned cell line (R278.5) from a rhesus monkey blastocyst that remains undifferentiated in continuous passage for > 1 year, maintains a normal XY karyotype, and expresses the cell surface markers (alkaline phosphatase, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81) that are characteristic of human embryonal carcinoma cells. R278.5 cells remain undifferentiated when grown on mouse embryonic fibroblast feeder layers but differentiate or die in the absence of fibroblasts, despite the presence of recombinant human leukemia inhibitory factor. R278.5 cells allowed to differentiate in vitro secrete bioactive chorionic gonadotropin into the medium, express chorionic gonadotropin alpha- and beta-subunit mRNAs, and express alpha-fetoprotein mRNA, indicating trophoblast and endoderm differentiation. When injected into severe combined immunodeficient mice, R278.5 cells consistently differentiate into derivatives of all three embryonic germ layers. These results define R278.5 cells as an embryonic stem cell line, to our knowledge, the first to be derived from any primate species.

The human embryo is not a feasible experimental system for the detailed study of implantation and early placentation, so surrogate systems have been sought for investigating the determination of the trophectoderm lineage, its differentiation into trophoblasts of the early implantation site, and subsequently the morphogenesis of the definitive placenta. An alternative to the use of embryos for studying early placental development was revealed by work with human embryonic stem cells (hESC), demonstrating BMP2/4-stimulated trophoblast differentiation, and spontaneous formation from embryoid bodies (EBs). These cells display a trophoblastic transcriptome, as well as a placental protein and steroid hormone secretory profile, and invasive and chemotactic behavior resembling human placental trophoblasts. With EB-derived trophoblasts, two-dimensional and three-dimensional paradigms and other modifications of the culture environment, including extracellular matrix and aggregation with placental fibroblasts, impact on trophoblast differentiation. Recent studies have questioned the identity of the trophoblasts directed by BMP treatment of hESC, and careful attention to culture conditions is needed to interpret different results among research groups. Although the precise placental counterpart of the hESC-derived trophoblast remains unclear, hESC-derived trophoblasts remain an intriguing platform for modeling early implantation.

International audience; Workshops are an important part of the IFPA annual meeting as they allow for discussion ofspecialised topics. At IFPA meeting 2011 there were twelve themed workshops, five of which aresummarized in this report. These workshops related to various aspects of placental biology: 1)immunology; 2) epigenetics; 3) comparative placentation; 4) trophoblast differentiation; 5) stemcells.

Type 5 acid phosphatase (ACP5; also known as tartrate-resistant acid phosphatase or uteroferrin) is a metalloprotein secreted by the endometrial glandular epithelium of pigs, mares, sheep, and water buffalo. In this paper, we describe the phylogenetic distribution of endometrial expression of ACP5 and demonstrate that endometrial expression arose early in evolution (i.e., before divergence of prototherian and therian mammals ∼166 million years ago). To determine expression of ACP5 in the pregnant endometrium, RNA was isolated from rhesus, mouse, rat, dog, sheep, cow, horse, armadillo, opossum, and duck-billed platypus. Results from RT-PCR and RNA-Seq experiments confirmed that ACP5 is expressed in all species examined. ACP5 was also demonstrated immunochemically in endometrium of rhesus, marmoset, sheep, cow, goat, and opossum. Alignment of inferred amino acid sequences shows a high conservation of ACP5 throughout speciation, with species-specific differences most extensive in the N-terminal and C-terminal regions of the protein. Analysis by Selecton indicated that most of the sites in ACP5 are undergoing purifying selection, and no sites undergoing positive selection were found. In conclusion, endometrial expression of ACP5 is a common feature in all orders of mammals and has been subjected to purifying selection. Expression of ACP5 in the uterus predates the divergence of therians and prototherians. ACP5 is an evolutionary conserved gene that likely exerts a common function important for pregnancy in mammals using a wide range of reproductive strategies.