Trophoblast inclusions and adverse birth outcomes.

OBJECTIVE: Trophoblast inclusions-cross sections of abnormal trophoblast bilayer infoldings-have previously been associated with aneuploidy, placenta accreta, and prematurity. This study was conducted to establish the relationship between trophoblast inclusions and a range of placental, pregnancy, and birth outcomes in a patient population with high smoking and alcohol exposure. Specifically, we sought to evaluate the association between the presence of trophoblast inclusions and 1) three primary birth outcomes: full-term birth, preterm birth, and stillbirth; 2) gestational age at delivery; and 3) specific placental pathologies. METHODS: Two slides containing chorionic villi were evaluated from 589 placentas that were collected from Stellenbosch University in Cape Town, South Africa as part of the prospective, multicenter cohort Safe Passage Study of the Prenatal Alcohol and SIDS and Stillbirth Network. The subsample included 307 full-term live births, 212 preterm live births, and 70 stillbirths. RESULTS: We found that the odds of identifying at least one trophoblast inclusion across two slides of chorionic villi was significantly higher for placentas from preterm compared to term liveborn deliveries (OR = 1.74; 95% CI: 1.22, 2.49, p = 0.002), with an even greater odds ratio for placentas from stillborn compared to term liveborn deliveries (OR = 4.95; 95% CI: 2.78, 8.80, p < 0.001). Gestational age at delivery was inversely associated with trophoblast inclusion frequency. Trophoblast inclusions were significantly associated with small for gestational age birthweight, induction of labor, villous edema, placental infarction, and inflammation of the chorionic plate. CONCLUSIONS: The novel associations that we report warrant further investigation in order to understand the complex network of biological mechanisms through which the factors that lead to trophoblast inclusions may influence or reflect the trajectory and health of a pregnancy. Ultimately, this line of research may provide critical insights that could inform both clinical and research applications.

YAP1 nuclear efflux and transcriptional reprograming follow membrane diminution upon VSV-G-induced cell fusion.

Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.

Maternal respiratory SARS-CoV-2 infection in pregnancy is associated with a robust inflammatory response at the maternal-fetal interface.

BACKGROUND: Pregnant women are at increased risk for severe outcomes from coronavirus disease 2019 (COVID-19), but the pathophysiology underlying this increased morbidity and its potential effect on the developing fetus is not well understood. METHODS: We assessed placental histology, ACE2 expression, and viral and immune dynamics at the term placenta in pregnant women with and without respiratory severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. FINDINGS: The majority (13 of 15) of placentas analyzed had no detectable viral RNA. ACE2 was detected by immunohistochemistry in syncytiotrophoblast cells of the normal placenta during early pregnancy but was rarely seen in healthy placentas at full term, suggesting that low ACE2 expression may protect the term placenta from viral infection. Using immortalized cell lines and primary isolated placental cells, we found that cytotrophoblasts, the trophoblast stem cells and precursors to syncytiotrophoblasts, rather than syncytiotrophoblasts or Hofbauer cells, are most vulnerable to SARS-CoV-2 infection in vitro. To better understand potential immune mechanisms shielding placental cells from infection in vivo, we performed bulk and single-cell transcriptomics analyses and found that the maternal-fetal interface of SARS-CoV-2-infected women exhibited robust immune responses, including increased activation of natural killer (NK) and T cells, increased expression of interferon-related genes, as well as markers associated with pregnancy complications such as preeclampsia. CONCLUSIONS: SARS-CoV-2 infection in late pregnancy is associated with immune activation at the maternal-fetal interface even in the absence of detectable local viral invasion. FUNDING: NIH (T32GM007205, F30HD093350, K23MH118999, R01AI157488, U01DA040588) and Fast Grant funding support from Emergent Ventures at the Mercatus Center.

SARS-CoV-2 infection in pregnancy is associated with robust inflammatory response at the maternal-fetal interface.

Pregnant women appear to be at increased risk for severe outcomes associated with COVID-19, but the pathophysiology underlying this increased morbidity and its potential impact on the developing fetus is not well understood. In this study of pregnant women with and without COVID-19, we assessed viral and immune dynamics at the placenta during maternal SARS-CoV-2 infection. Amongst uninfected women, ACE2 was detected by immunohistochemistry in syncytiotrophoblast cells of the normal placenta during early pregnancy but was rarely seen in healthy placentas at full term. Term placentas from women infected with SARS-CoV-2, however, displayed a significant increase in ACE2 levels. Using immortalized cell lines and primary isolated placental cells, we determined the vulnerability of various placental cell types to direct infection by SARS-CoV-2 in vitro. Yet, despite the susceptibility of placental cells to SARS-CoV-2 infection, viral RNA was detected in the placentas of only a subset (~13%) of women in this cohort. Through single cell transcriptomic analyses, we found that the maternal-fetal interface of SARS-CoV-2-infected women exhibited markers associated with pregnancy complications, such as preeclampsia, and robust immune responses, including increased activation of placental NK and T cells and increased expression of interferon-related genes. Overall, this study suggests that SARS-CoV-2 is associated with immune activation at the maternal-fetal interface even in the absence of detectable local viral invasion. While this likely represents a protective mechanism shielding the placenta from infection, inflammatory changes in the placenta may also contribute to poor pregnancy outcomes and thus warrant further investigation.

Trophoblast inclusions in the human placenta: Identification, characterization, quantification, and interrelations of subtypes.

We sought to examine placentas enriched for trophoblast inclusions (TIs) in order to characterize, quantify, and examine the interrelations between subtypes of TIs to better understand their underlying biology. We examined a cohort of 600 placentas from deliveries between 200 and 430 weeks of gestation. Forty-five percent of the placentas had at least one TI in the two slides examined. Four percent of the placentas had 10 or more TIs and two placentas had more than 70 TIs. Four distinct TI subtypes were observed: inclusionoids (early forming inclusions), inclusions, calcified inclusions, and calcified bodies. We suggest this reflects a developmental trajectory of TI maturation, the timing of which might be useful when comparing TI expression to clinical outcomes.

An experimental test of the ovulatory homolog model of female orgasm.

The ovulatory homolog model of female orgasm posits that the neuro-endocrine mechanisms underlying female orgasm evolved from and are homologous to the mechanisms mediating copulation-induced ovulation in some mammals. This model predicts that pharmacological agents that affect human orgasm, such as fluoxetine, should also affect ovulation in animals with copulation-induced ovulation, such as rabbits. We tested this prediction by treating rabbits with daily doses of fluoxetine for 2 wk and found that fluoxetine treatment reduces the number of ovulations postcopulation by 30%. In a second experiment we tested whether this result was mediated by an effect on the brain or via peripheral serotonin functions. We treated animals with fluoxetine and induced ovulation with a single injection of human chorionic gonadotropin. In this experiment ovulation rate was nominally reduced by only 8%, which is statistically not significant. We conclude that the effect of fluoxetine on copulation-induced ovulation rate supports the ovulatory homolog model of female orgasm, suggesting that female orgasm has very deep evolutionary roots among the early eutherian mammals.

Pathway of Maternal Serotonin to the Human Embryo and Fetus.

Serotonin [5-hydroxytryptamine (5-HT)] is essential to intrauterine development, but its source is debated. We used immunocytochemistry to gauge 5-HT, its biosynthetic enzyme tryptophan hydroxylase 1 (TPH1); an importer (serotonin transporter, 5-HTT/SERT/SLC6A); other transporters [P-glycoprotein 1 (P-gp/ABCB1), OCT3/SLC22A3, and gap junction connexin-43]; and the 5-HT degradative enzyme monoamine oxidase A (MAOA) in sections of placentas. In humans, 5-HT was faintly stained only in first-trimester trophoblasts, whereas TPH1 was not seen at any stage. SERT was expressed in syncytiotrophoblasts and, more strongly, in cytotrophoblasts. MAOA was prominent in syncytiotrophoblasts, OCT3 and gap junctions were stained in cytotrophoblasts, and P-gp was present at the apical surfaces of both epithelia. 5-HT added to cultured placental explants accumulated in the trophoblast epithelium and reached the villus core vessels. Trophoblast uptake was blocked by the SERT inhibitor escitalopram. Inhibition of gap junctions with heptanol prevented the accumulation of 5-HT in cytotrophoblasts, whereas blocking OCT3 with decynium-22 and P-gp with mitotane led to its accumulation in cytotrophoblasts. Reducing 5-HT destruction by inhibiting MAOA with clorgyline increased the accumulation of 5-HT throughout the villus. In the mouse fetus, intravascular platelets stained prominently for 5-HT at day 13.5, whereas the placenta and yolk sac endoderm were both negative. TPH1 was not detected, but SERT was prominent in these mouse tissues. We conclude that serotonin is conveyed from the maternal blood stream through syncytiotrophoblasts, cytotrophoblasts and the villus core to the fetus through a physiological pathway that involves at least SERT, gap junctions, P-gp, OCT3, and MAOA.

Type I interferons instigate fetal demise after Zika virus infection.

Zika virus (ZIKV) infection during pregnancy is associated with adverse fetal outcomes, including microcephaly, growth restriction, and fetal demise. Type I interferons (IFNs) are essential for host resistance against ZIKV, and IFN-α/ß receptor (IFNAR)-deficient mice are highly susceptible to ZIKV infection. Severe fetal growth restriction with placental damage and fetal resorption is observed after ZIKV infection of type I IFN receptor knockout (Ifnar1-/-) dams mated with wild-type sires, resulting in fetuses with functional type I IFN signaling. The role of type I IFNs in limiting or mediating ZIKV disease within this congenital infection model remains unknown. In this study, we challenged Ifnar1-/- dams mated with Ifnar1+/- sires with ZIKV. This breeding scheme enabled us to examine pregnant dams that carry a mixture of fetuses that express (Ifnar1+/-) or do not express IFNAR (Ifnar1-/-) within the same uterus. Virus replicated to a higher titer in the placenta of Ifnar1-/- than within the Ifnar1+/- concepti. Yet, rather unexpectedly, we found that only Ifnar1+/- fetuses were resorbed after ZIKV infection during early pregnancy, whereas their Ifnar1-/- littermates continue to develop. Analyses of the fetus and placenta revealed that, after ZIKV infection, IFNAR signaling in the conceptus inhibits development of the placental labyrinth, resulting in abnormal architecture of the maternal-fetal barrier. Exposure of midgestation human chorionic villous explants to type I IFN, but not type III IFNs, altered placental morphology and induced cytoskeletal rearrangements within the villous core. Our results implicate type I IFNs as a possible mediator of pregnancy complications, including spontaneous abortions and growth restriction, in the context of congenital viral infections.

Trophoblast inclusions are significantly increased in the placentas of children in families at risk for autism.

BACKGROUND: Gestation is a critical window for neurodevelopmental vulnerability. This study examined whether the presence of trophoblast inclusions (TIs) in the placenta could serve as a predictor for children at elevated risk for autism spectrum disorder (ASD). METHODS: Placentas were obtained from 117 births in the MARBLES (Markers of Autism Risk in Babies-Learning Early Signs) cohort of families who have one or more previous biological children with ASD, placing their newborn at elevated risk for neurodevelopmental compromise. Control samples were obtained from 100 uncomplicated term pregnancies of multiparous women with one or more typically developing biological children. Frequency of TIs was compared across the two groups. RESULTS: Placentas from at-risk pregnancies had an eightfold increased odds of having two or more TIs compared with control samples (odds ratio: 8.0, 95% confidence interval: 3.6-18.0). The presence of≥2 TIs yielded a sensitivity of 41% and a specificity of 92% for predicting ASD risk status, whereas≥4 TIs yielded a sensitivity of 19%, a specificity of 99.9%, and a positive likelihood ratio of 242 and conservatively predicted an infant with a 74% probability of being at risk for ASD. CONCLUSIONS: Our findings suggest that the placentas from women whose fetuses are at elevated risk for autism are markedly different from control placentas. These differences are manifested histologically as TIs. Their identification has the possibility of identifying newborns at risk for ASD who might benefit from targeted early interventions aimed at preventing or ameliorating behavioral symptoms and optimizing developmental outcomes.