Atypical initial cleavage patterns minimally impact rhesus macaque in vitro embryo morphokinetics and embryo outgrowth development†.

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 promise of placental extracellular vesicles: models and challenges for diagnosing placental dysfunction in utero†.

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.

Embryotoxic impact of Zika virus in a rhesus macaque in vitro implantation model†.

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.

Zika virus impacts extracellular vesicle composition and cellular gene expression in macaque early gestation trophoblasts.

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.

Placenta-derived macaque trophoblast stem cells: differentiation to syncytiotrophoblasts and extravillous trophoblasts reveals phenotypic reprogramming.

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.

Defining the rhesus macaque placental miRNAome: Conservation of expression of placental miRNA clusters between the macaque and human.

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.

Ocular and uteroplacental pathology in a macaque pregnancy with congenital Zika virus infection.

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.