Neuropilin-1 (NRP-1) is an essential regulator of maternal immune tolerance, placentation, and angiogenesis. Its dysregulation in preeclampsia (PE) and human immunodeficiency virus (HIV) infection implicates NRP-1 in disease susceptibility and progression. Therefore, this study investigates placental NRP-1 immunoexpression in HIV-complicated preeclamptic pregnancies in South African women of African ancestry receiving antiretroviral therapy. Immunohistochemistry of recombinant anti-neuropilin-1 antibody was performed on placental tissue from 30 normotensive and 60 early onset (EOPE) and late-onset (LOPE) preeclamptic women stratified by HIV status. Qualitative analysis of NRP-1 immunostaining within the chorionic villi revealed a predominant localization in trophoblasts and syncytial knots as well as endothelial, fibroblast-like, and Hofbauer cells. Following morphometric evaluation, we report that PE and HIV infection and/or antiretroviral usage independently downregulate placental NRP-1 immunoexpression; however, as a comorbidity, this decline is further augmented within the conducting and exchange villi. Furthermore, reduced immunoexpression of NRP-1 in EOPE compared with LOPE villi may be due to maternal-fetal maladaptation. It is plausible that the decreased NRP-1 immunoexpression in PE placentae facilitates syncytiotrophoblast apoptosis and subsequent deportation of NRP-1 into the maternal circulation, contributing to the anti-angiogenic milieu of PE. We hypothesize that the intense NRP-1 immunoreactivity observed in Hofbauer cells at the maternal-fetal interface may contribute to the natural prevention mechanism of HIV vertical transmission.

Damage to the genome can accelerate aging. The percentage of aneuploid cells, that is, cells with an abnormal number of chromosomes, increases during aging; however, it is not clear whether increased aneuploidy accelerates aging. Here, we report an individual showing premature aging phenotypes of various organs including early hair loss, atrophic skin, and loss of hematopoietic stem cells; instability of chromosome numbers known as mosaic variegated aneuploidy (MVA); and spindle assembly checkpoint (SAC) failure. Exome sequencing identified a de novo heterozygous germline missense mutation of c.856C>A (p.R286S) in the mitotic activator CDC20. The mutant CDC20 showed lower binding affinity to BUBR1 during the formation of the mitotic checkpoint complex (MCC), but not during the interaction between MCC and the anaphase-promoting complex/cyclosome (APC/C)-CDC20 complex. While heterozygous knockout of CDC20 did not induce SAC failure, knock-in of the mutant CDC20 induced SAC failure and random aneuploidy in cultured cells, indicating that the particular missense mutation is pathogenic probably via the resultant imbalance between MCC and APC/C-CDC20 complex. We postulate that accelerated chromosome number instability induces premature aging in humans, which may be associated with early loss of stem cells. These findings could form the basis of a novel disease model of the aging of the body and organs.

Watasenia scintillans, a sparkling enope squid, has bioluminescence organs to illuminate its body with its own luciferase activity. To clarify the molecular mechanism underlying its scintillation, we analysed high-throughput sequencing data acquired previously and obtained draft genome sequences accomplished with comparative genomic data among the cephalopods. The genome mapped by transcriptome data showed that (1) RNA editing contributed to transcriptome variation of lineage specific genes, such as W. scintillans luciferase, and (2) two types of luciferase enzymes were characterized with reasonable 3D models docked to a luciferin molecule. We report two different types of luciferase in one organism and possibly related to variety of colour types in the W. scintillans fluorescent organs.

Zika virus (ZIKV) infection during pregnancy leads to devastating fetal outcomes, including intrauterine growth restriction and microcephaly. Greater understanding of mechanisms underlying ZIKV maternal-fetal transmission is needed to develop new therapeutic interventions. Here, we define an important role for the autophagy pathway in ZIKV vertical transmission. ZIKV infection induced autophagic activity in human trophoblasts and pharmacological inhibition limited ZIKV infectivity. Furthermore, deficiency in an essential autophagy gene, Atg16l1, in mice limited ZIKV vertical transmission and placental and fetal damage and overall improved placental and fetal outcomes. This protection was due to a placental trophoblast cell-autonomous effect of autophagic activity, not to alterations in systemic maternal ZIKV infection. Finally, an autophagy inhibitor, hydroxychloroquine, approved for use in pregnant women, attenuated placental and fetal ZIKV infection and ameliorated adverse placental and fetal outcomes. Our study reveals new insights into the mechanism of ZIKV vertical transmission and suggests that an autophagy-based therapeutic warrants possible evaluation in humans to diminish the risks of ZIKV maternal-fetal transmission.

Emerging evidence indicates that circular RNA (circRNA) and N6-methyladenosine (m6A) play critical roles in cervical cancer. However, the synergistic effect of circRNA and m6A on cervical cancer progression is unclear. In the present study, our sequencing data revealed that a novel m6A-modified circRNA (circARHGAP12, hsa_circ_0000231) upregulated in the cervical cancer tissue and cells. Interestingly, the m6A modification of circARHGAP12 could amplify its enrichment. Functional experiments illustrated that circARHGAP12 promoted the tumor progression of cervical cancer in vivo and vitro. Furthermore, MeRIP-Seq illustrated that there was a remarkable m6A site in FOXM1 mRNA. CircARHGAP12 interacted with m6A reader IGF2BP2 to combine with FOXM1 mRNA, thereby accelerating the stability of FOXM1 mRNA. In conclusion, we found that circARHGAP12 exerted the oncogenic role in cervical cancer progression through m6A-dependent IGF2BP2/FOXM1 pathway. These findings may provide new concepts for cervical cancer biology and pathological physiology.

Geometry of the placental villous vasculature is a key determinant of maternal-fetal nutrient exchange for optimal fetal growth. Recent advances in tissue clarification techniques allow for deep high-resolution imaging with confocal microscopy; however, the methodology lacks a signal:noise ratio of sufficient magnitude to allow for quantitative analysis. Thus, we sought to develop a reproducible method to investigate the 3D vasculature of the nonhuman primate placenta for subsequent data analysis. Fresh placental tissue was dissected, formalin fixed, clarified using a modified Visikol® protocol and immunolabeled for CD31 (fetal endothelium) and cytokeratin-7 (villous trophoblast) for confocal imaging of the microanatomy. We present a detailed clarification and staining protocol augmented for imaging of nonhuman primate placental tissue. The image stacks generated by this refined staining method and our data acquisition parameters can be analyzed quantitatively to provide insights regarding the villous and vascular micro-anatomy of the placenta.

The placenta forms a maternal-fetal junction that supports many physiological functions such as the supply of nutrition and exchange of gases and wastes. Establishing an in vitro culture model of human and non-human primate trophoblast stem/progenitor cells is important for investigating the process of early placental development and trophoblast differentiation. In this study, we have established five trophoblast stem cell (TSC) lines from cynomolgus monkey blastocysts, named macTSC #1-5. Fibroblast growth factor 4 (FGF4) enhanced proliferation of macTSCs, while other exogenous factors were not required to maintain their undifferentiated state. macTSCs showed a trophoblastic gene expression profile and trophoblast-like DNA methylation status and also exhibited differentiation capacity towards invasive trophoblast cells and multinucleated syncytia. In a xenogeneic chimera assay, these stem cells contributed to trophectoderm (TE) development in the chimeric blastocysts. macTSC are the first primate trophoblast cell lines whose proliferation is promoted by FGF4. These cell lines provide a valuable in vitro culture model to analyze the similarities and differences in placental development between human and non-human primates.

The syncytiotrophoblast is a human epithelial cell that is bathed in maternal blood on the maternal-facing surface of the human placenta. It therefore acts as a barrier and exchange interface between the mother and fetus. Syncytiotrophoblast dysfunction is a feature of pregnancy pathologies, like preeclampsia. Dysfunctional syncytiotrophoblasts display a loss of microvilli, which is a marker of aberrant apical-basal polarization, but little data exist about the regulation of syncytiotrophoblast polarity. Atypical PKC isoforms are conserved polarity regulators. Thus, we hypothesized that aPKC isoforms regulate syncytiotrophoblast polarity. Using human placental explant culture and primary trophoblasts, we found that loss of aPKC activity or expression induces syncytiotrophoblast gasdermin-E-dependent pyroptosis, a form of programmed necrosis. We also establish that TNF-α induces an isoform-specific decrease in aPKC expression and gasdermin-E-dependent pyroptosis. Therefore, aPKCs are homeostatic regulators of the syncytiotrophoblast function and a pathogenically relevant pro-inflammatory cytokine leads to the induction of programmed necrosis at the maternal-fetal interface. Hence, our results have important implications for the pathobiology of placental disorders like preeclampsia.

Adapting the energy metabolism state to changing bioenergetic demands is essential for mammalian development accompanying massive cell proliferation and cell differentiation. However, it remains unclear how developing embryos meet the changing bioenergetic demands during the chorioallantoic branching (CB) stage, when the maternal-fetal exchange of gases and nutrients is promoted. In this study, using metabolome analysis with mass-labeled glucose, we found that developing embryos redirected glucose carbon flow into the pentose phosphate pathway via suppression of the key glycolytic enzymes PFK-1 and aldolase during CB. Concomitantly, embryos exhibited an increase in lactate pool size and in the fractional contribution of glycolysis to lactate biosynthesis. Imaging mass spectrometry visualized lactate-rich tissues, such as the dorsal or posterior neural tube, somites and head mesenchyme. Furthermore, we found that the heterochronic gene Lin28a could act as a regulator of the metabolic changes observed during CB. Perturbation of glucose metabolism rewiring by suppressing Lin28a downregulation resulted in perinatal lethality. Thus, our work demonstrates that developing embryos rewire glucose metabolism following CB for normal development.

Toxoplasma gondii is an obligate intracellular protozoan with worldwide distribution. Diagnosis of toxoplasmosis is a very critical issue, especially in pregnant women and immunocompromised patients. The aim of this study was rapid detection of T. gondii DNA in peripheral blood samples (PBS) employing HRM technique and using RE gene.

Although partial androgen insensitivity syndrome (PAIS) is caused by attenuated responsiveness to androgens, androgen receptor gene (AR) mutations on the coding regions and their splice sites have been identified only in <25% of patients with a diagnosis of PAIS. We performed extensive molecular studies including whole exome sequencing in a Japanese family with PAIS, identifying a deep intronic variant beyond the branch site at intron 6 of AR (NM_000044.4:c.2450-42 G > A). This variant created the splice acceptor motif that was accompanied by pyrimidine-rich sequence and two candidate branch sites. Consistent with this, reverse transcriptase (RT)-PCR experiments for cycloheximide-treated lymphoblastoid cell lines revealed a relatively large amount of aberrant mRNA produced by the newly created splice acceptor site and a relatively small amount of wildtype mRNA produced by the normal splice acceptor site. Furthermore, most of the aberrant mRNA was shown to undergo nonsense mediated decay (NMD) and, if a small amount of aberrant mRNA may have escaped NMD, such mRNA was predicted to generate a truncated AR protein missing some functional domains. These findings imply that the deep intronic mutation creating an alternative splice acceptor site resulted in the production of a relatively small amount of wildtype AR mRNA, leading to PAIS.

The human placenta plays a key role in reproduction and serves as a major interface for maternofetal exchange of nutrients. Study of human placenta pathology presents a great experimental challenge because it is not easily accessible. In this paper, a 3D placenta-on-a-chip model is developed by bioengineering techniques to simulate the placental interface between maternal and fetal blood in vitro. In this model, trophoblasts cells and human umbilical vein endothelial cells are cultured on the opposite sides of a porous polycarbonate membrane, which is sandwiched between two microfluidic channels. Glucose diffusion across this barrier is analyzed under shear flow conditions. Meanwhile, a numerical model of the 3D placenta-on-a-chip model is developed. Numerical results of concentration distributions and the convection-diffusion mass transport is compared to the results obtained from the experiments for validation. Finally, effects of flow rate and membrane porosity on glucose diffusion across the placental barrier are studied using the validated numerical model. The placental model developed here provides a potentially helpful tool to study a variety of other processes at the maternal-fetal interface, for example, effects of drugs or infections like malaria on transport of various substances across the placental barrier.

FBXW7 is a ubiquitin ligase that mediates ubiquitylation of oncoproteins, such as c-Myc, cyclin E, Notch and c-Jun. FBXW7 is a known tumor-suppressor gene, and mutations in FBXW7 have been reported in various human malignancies. In this study, we examined the sequences of the FBXW7 and p53 genes in 57 ovarian cancer clinical samples. Interestingly, we found no FBXW7 mutations associated with amino acid changes. We also investigated FBXW7 expression levels in 126 epithelial ovarian tumors. FBXW7 expression was negatively correlated with the malignant potential of ovarian tumors. That is to say, FBXW7 expression levels in ovarian cancer samples were significantly lower than those in borderline and benign tumors (P < 0.01). FBXW7 expression levels in serous carcinoma samples were the lowest among four major histological subtypes. In addition, p53-mutated ovarian cancer samples showed significantly lower levels of FBXW7 expression compared with p53 wild-type cancer samples (P < 0.001). DNA methylation arrays and bisulfite PCR sequencing experiments revealed that 5'-upstream regions of FBXW7 gene in p53-mutated samples were significantly higher methylated compared with those in p53 wild-type samples (P < 0.01). This data indicates that p53 mutations might suppress FBXW7 expression through DNA hypermethylation of FBXW7 5'-upstream regions. Thus, FBXW7 expression was downregulated in ovarian cancers, and was associated with p53 mutations and the DNA methylation status of the 5'-upstream regions of FBXW7.

The human placenta is a critical organ, mediating the exchange of nutrients, oxygen, and waste products between fetus and mother. Placental malaria (PM) resulted from Plasmodium falciparum infections causes up to 200 thousand newborn deaths annually, mainly due to low birth weight, as well as 10 thousand mother deaths. In this work, a placenta-on-a-chip model is developed to mimic the nutrient exchange between the fetus and mother under the influence of PM. In this model, trophoblasts cells (facing infected or uninfected blood simulating maternal blood and termed "trophoblast side") and human umbilical vein endothelial cells (facing uninfected blood simulating fetal blood and termed "endothelial" side) are cultured on the opposite sides of an extracellular matrix gel in a compartmental microfluidic system, forming a physiological barrier between the co-flow tubular structure to mimic a simplified maternal-fetal interface in placental villi. The influences of infected erythrocytes (IEs) sequestration through cytoadhesion to chondroitin sulfate A (CSA) expressed on the surface of trophoblast cells, a critical feature of PM, on glucose transfer efficiency across the placental barrier was studied. To create glucose gradients across the barrier, uninfected erythrocyte or IE suspension with a higher glucose concentration was introduced into the "trophoblast side" and a culture medium with lower glucose concentration was introduced into the "endothelial side". The glucose levels in the endothelial channel in response to CSA-adherent erythrocytes infected with CS2 line of parasites in trophoblast channel under flow conditions was monitored. Uninfected erythrocytes served as a negative control. The results demonstrated that CSA-binding IEs added resistance to the simulated placental barrier for glucose perfusion and decreased the glucose transfer across this barrier. The results of this study can be used for better understanding of PM pathology and development of models useful in studying potential treatment of PM.

Fetal growth and newborn weight from ovine gestations at high altitudes (HA) are greater in ewes that live at HA for several generations than in those native to low altitudes (LA) exposed to HA only during pregnancy. Because the placenta is a key regulator of fetal growth, the present study compared placental characteristics in term pregnancies among ewes native to HA and LA. Conception occurred at HA and ewes continued to reside at HA throughout pregnancy or conception occurred at LA and ewes were transported to HA or remained at LA (controls). Ewes native to LA were moved to HA shortly after mating (group LH) and joined with pregnant ewes native to HA (group HH). After parturition, placental cotyledons were counted and measured for total area and histological estimation of surface occupied by vasculature. The total surface of the cotyledons and surface occupied by vasculature were greater at HA, whereas the number of cotyledons was smaller at HA. These changes were more pronounced in ewes of the HH compared with the LH group. The present study showed that exposure to HA induces, in pregnant ewes, placental morphological changes that may improve maternal-fetal exchange. Moreover, because of accentuation of placental changes in ewes with long-term residence at HA, this appears to be an efficient mechanism of adaptation to hypobaric hypoxia.

In the human placenta, two trophoblast cell layers separate the maternal blood from the villous basement membrane and fetal capillary endothelial cells. The inner layer, which is complete early in pregnancy and later becomes discontinuous, comprises the proliferative mononuclear cytotrophoblasts, which fuse together and differentiate to form the outer layer of multinucleated syncytiotrophoblasts. Because the syncytiotrophoblasts are responsible for key maternal-fetal exchange functions, tight regulation of this differentiation process is critical for the proper development and the functional role of the placenta. The molecular mechanisms regulating the fusion and differentiation of trophoblasts during human pregnancy remain poorly understood. To decipher the interactions of non-coding RNAs (ncRNAs) in this process, we exposed cultured primary human trophoblasts to standard in vitro differentiation conditions or to conditions known to hinder this differentiation process, namely exposure to hypoxia (O2 < 1%) or to the addition of dimethyl sulfoxide (DMSO, 1.5%) to the culture medium. Using next generation sequencing technology, we analyzed the differential expression of trophoblastic lncRNAs, miRNAs, and mRNAs that are concordantly modulated by both hypoxia and DMSO. Additionally, we developed a model to construct a lncRNA-miRNA-mRNA co-expression network and inferred the functions of lncRNAs and miRNAs via indirect gene ontology analysis. This study improves our knowledge of the interactions between ncRNAs and mRNAs during trophoblast differentiation and identifies key biological processes that may be impaired in common gestational diseases, such as fetal growth restriction or preeclampsia.

Physical activity (PA) has beneficial effects on the function of many organs by modulating their vascular development. Regular PA during pregnancy is associated with favorable short- and long-term outcomes for both mother and fetus. During pregnancy, appropriate vascularization of the placenta is crucial for adequate maternal-fetal nutrient and gas exchange. How PA modulates angiogenic factors, VEGF, and its receptors in the human placenta, is as of yet, unknown. We objectively measured the PA of women at 24-28 and 34-38 weeks of gestation. Participants were considered "active" if they had met or exceeded 150 min of moderate-intensity PA per week during their 2nd trimester. Term placenta tissues were collected from active (n = 23) or inactive (n = 22) women immediately after delivery. We examined the expression of the angiogenic factors VEGF, PlGF, VEGFR-1, and VEGFR-2 in the placenta. Western blot analysis showed VEGF and its receptor, VEGFR-1 was significantly (p < 0.05) higher both at the protein and mRNA levels in placenta from physically active compared to inactive women. No difference in VEGFR-2 was observed. Furthermore, immunohistochemistry showed differential staining patterns of VEGF and its receptors in placental endothelial, stromal, and trophoblast cells and in the syncytial brush border. In comparison, PlGF expression did not differ either at the protein or mRNA level in the placenta from physically active or inactive women. The expression and localization pattern of VEGF and its receptors suggest that PA during pregnancy may support a pro-angiogenic milieu to the placental vascular network.

Tristetraprolin (TTP) regulates the stability of multiple targets that have important biological roles. However, the role of TTP in trophoblasts at the maternal-fetal interface remains poorly understood. We demonstrated that TTP was upregulated in placental trophoblasts from patients with recurrent miscarriages (RMs). Immunofluorescence and immunoblotting analyses indicated that TTP was redistributed from the nucleus to the cytoplasm in trophoblasts from patients with RMs. Trophoblast invasion and proliferation was repressed by TTP overexpression and was enhanced by TTP knockdown. Interestingly, TTP knockdown promoted trophoblast invasion in an ex vivo explant culture model. Furthermore, TTP overexpression in trophoblasts significantly inhibited the expression of the long non-coding RNA (lncRNA) HOTAIR. TTP was found to regulate HOTAIR expression by a posttranscriptional mechanism. To RNA immunoprecipitation (RIP) and RNA-protein, pull-down identified TTP as a specific binding partner that decreased the half-life of HOTAIR and lowered steady-state HOTAIR expression levels, indicating a novel posttranscriptional regulatory mechanism. Our findings identify a novel function for TTP in lncRNA regulation and provide important insights into the regulation of lncRNA expression. This study reveals a new pathway governing the regulation of TTP/HOTAIR in trophoblast cell invasion during early pregnancy.

Human cytomegalovirus (CMV) is a highly prevalent herpesvirus that is often transmitted to the neonate via breast milk. Postnatal CMV transmission can have negative health consequences for preterm and immunocompromised infants, but any effects on healthy term infants are thought to be benign. Furthermore, the impact of CMV on the composition of the hundreds of bioactive factors in human milk has not been tested. Here, we utilize a cohort of exclusively breastfeeding full term mother-infant pairs to test for differences in the milk transcriptome and metabolome associated with CMV, and the impact of CMV in breast milk on the infant gut microbiome and infant growth. We find upregulation of the indoleamine 2,3- dioxygenase (IDO) tryptophan-to-kynurenine metabolic pathway in CMV+ milk samples, and that CMV+ milk is associated with decreased Bifidobacterium in the infant gut. Our data indicate a complex relationship between milk CMV, milk kynurenine, and infant growth; with kynurenine positively correlated, and CMV viral load negatively correlated, with infant weight-for-length at 1 month of age. These results suggest CMV transmission, CMV-related changes in milk composition, or both may be modulators of full term infant development.

The human placental barrier facilitates many key functions during pregnancy, most notably the exchange of all substances between the mother and fetus. However, preclinical models of the placental barrier often lacked the multiple cell layers, syncytialization of the trophoblast cells and the low oxygen levels that are present within the body. Therefore, we aimed to design and develop an in vitro model of the placental barrier that would reinstate these factors and enable improved investigations of barrier function. BeWo placental trophoblastic cells and human umbilical vein endothelial cells were co-cultured on contralateral sides of an extracellular matrix-coated transwell insert to establish a multilayered barrier. Epidermal growth factor and forskolin led to significantly increased multi-nucleation of the BeWo cell layer and increased biochemical markers of syncytial fusion, for example syncytin-1 and hCGβ. Our in vitro placental barrier possessed size-specific permeability, with 4000-Da molecules experiencing greater transport and a lower apparent permeability coefficient than 70 000-Da molecules. We further demonstrated that the BeWo layer had greater resistance to smaller molecules compared to the endothelial layer. Chronic, physiologically low oxygen exposure (3-8%) increased the expression of hypoxia-inducible factor 1α and syncytin-1, further increased multi-nucleation of the BeWo cell layer and decreased barrier permeability only against smaller molecules (457 Da/4000 Da). In conclusion, we built a novel in vitro co-culture model of the placental barrier that possessed size-specific permeability and could function under physiologically low oxygen levels. Importantly, this will enable future researchers to better study the maternal-fetal transport of nutrients and drugs during pregnancy.