ABSTRACT
Problem: Although it is rare for a SARS-CoV-2 infection to transmit vertically to the fetus during pregnancy, there is a significantly increased risk of adverse pregnancy outcomes due to maternalCOVID- 19. However, there is a poor understanding of such risks because mechanistic studies on how SARS-CoV-2 infection disrupts placental homeostasis are significantly lacking. The SARS-CoV-2 proteome includes multiple structural and non-structural proteins, including the non-structural accessory proteinORF3a. The roles of these proteins in mediating placental infection remain undefined. We and others have shown that autophagy activity in placental syncytium is essential for barrier function and integrity. Here, we have used clinical samples and cultured trophoblast cells to evaluate syncytial integrity of placenta exposed to SARS-CoV-2. The objective of our study was to investigate potential mechanisms through which SARS-CoV-2 impairs placental homeostasis and causes adverse pregnancy outcomes. We tested the central hypothesis that an essential SARS-CoV-2 non-structural and accessory protein, ORF3a, uniquely (amongst multiple viral proteins tested) with a novel three-dimensional structure andwith no homology to any other proteins is a key modulator of placental trophoblast cell dynamics via autophagy and intracellular trafficking of a tight junction protein (TJP), ZO-1. Method(s): We used clinical samples and cultured trophoblast cells to evaluate syncytial integrity of placentas exposed to SARS-CoV- 2. Autophagic flux was measured in placental villous biopsies from SARS-CoV-2-exposed and unexposed pregnant women by quantifying the expression of autophagy markers, LC3 and P62. Trophoblast cells (JEG-3, Forskolin-treated JEG-3, HTR8/SVneo, or primary human trophoblasts (PHTs)) were transfected with expression plasmids encoding SARS-CoV-2 proteins including ORF3a. Using western blotting, multi-label immunofluorescence, and confocal imaging, we analyzed the effect of ORF3a on the autophagy, differentiation, invasion, and intracellular trafficking of ZO-1 in trophoblasts. Using coimmunoprecipitation assays, we tested ORF3a interactions with host proteins. t-tests and one-way analyses of variance (ANOVAs) with post hoc tests were used to assess the data, with significance set at P < .05. Result(s): We discovered :1) increased activation of autophagy, but incomplete processing of autophagosome-lysosomal degradation;2) accumulation of protein aggregates in placentas exposed to SARS-CoV- 2. Mechanistically, we showed that the SARS-CoV-2 ORF3a protein, uniquely 3) blocks the autophagy-lysosomal degradation process;4) inhibits maturation of cytotrophoblasts into syncytiotrophoblasts (STBs);5) reduces production ofHCG-beta, a key pregnancy hormone that is also essential for STB maturation;and 6) inhibits trophoblast invasive capacity. Furthermore, ORF3a harbors an intrinsically disordered C-terminus withPDZ-bindingmotifs.We show for the first time that, 7) ORF3a binds to and co-localizes with the PDZ domain of ZO-1, a junctional protein that is essential for STB maturation and the integrity of the placental barrier. Conclusion(s): Our work outlines a new molecular and cellular mechanism involving the SARS-CoV-2 accessory protein ORF3a that may drive the virus's ability to infect the placenta and damage placental syncytial integrity. This implies that the mechanisms facilitating viral maturation, such as the interaction of ORF3a with host factors, can be investigated for additional functionality and even targeted for developing new intervention strategies for treatment or prevention of SARS-CoV-2 infection at the maternal-fetal interface.
ABSTRACT
Introduction: Starvation ketoacidosis represents one of the three metabolic acidoses caused by the accumulation of ketone bodies within the bloodstream. Outside of late pregnancy, it is a relatively rare condition. In late pregnancy, the placental production of the hormones estrogen, cortisol, and human placental lactogen combined with increased lipolysis causes greater insulin resistance and an overall catabolic state which improves nutrient availability for vital fetal growth. However, this also allows for a magnified response to fasting that results in increased ketone production and in rare cases “accelerated starvation.” In this case, we present a 25-year-old pregnant patient who presented with nausea, vomiting, and poor oral intake, who was found to be in starvation ketoacidosis. Case Description: A 25-year-old G2P1001 cis female with a previous medical history of migraines presented at 33 weeks gestation with nausea, vomiting, and poor oral intake for four days prior to admission in the setting of COVID-19 infection. Patient presented hemodynamically stable and in no acute distress. Fetal non stress test on admission was reactive. Initial lab work revealed a glucose of 95, anion gap of 21, and a bicarbonate level of 7. A beta hydroxybutyrate (BHB) level was elevated at 5.26. Arterial blood gas showed a pH of 7.2 and a PCO2 of 23, consistent with an anion gap metabolic acidosis. Urinalysis revealed 3+ ketones. Overall labs were consistent with starvation ketoacidosis and the patient was immediately resuscitated with dextrose containing intravenous fluids and an insulin drip to help shunt away from ketoacidosis. Her BHB rapidly downtrended to 1.28 within 12 hours and within 24 hours it normalized. Her metabolic acidosis continued to improve throughout her hospitalization. She was able to tolerate a regular diet prior to being discharged home. A few weeks later, she had an uncomplicated full term delivery of a healthy baby. Discussion: Starvation ketoacidosis outside of pregnancy is rare and takes at least two weeks to manifest as a mild ketoacidosis. In pregnancy, patients are in an insulin resistant state which increases with gestational age, making them prone to ketoacidosis particularly in the second and third trimesters. Ketoacidosis in pregnancy is not only harmful for the pregnant individual, but for the developing fetus as well. Ketones can cross the placental barrier, leading to neurological impairment and even fetal demise if the acidosis is not addressed quickly. Prompt treatment with IV fluids, dextrose, and insulin is imperative to prevent neurodevelopmental compromise. Patients with appropriate and timely treatment can continue on to have uncomplicated pregnancies and deliveries.