COVID-19 Infection in Pregnancy: PCR Cycle Thresholds, Placental Pathology, and Perinatal Outcomes.

(1) This study aimed to evaluate characteristics, perinatal outcomes, and placental pathology of pregnant women with or without SARS-CoV-2 infection in the context of maternal PCR cycle threshold (CT) values. (2) This was a retrospective case-control study in a third-level health center in Mexico City with universal screening by RT-qPCR. The association of COVID-19 manifestations, preeclampsia, and preterm birth with maternal variables and CT values were assessed by logistic regression models and decision trees. (3) Accordingly, 828 and 298 women had a negative and positive test, respectively. Of those positive, only 2.6% of them presented mild to moderate symptoms. Clinical characteristics between both groups of women were similar. No associations between CT values were found for maternal features, such as pre-gestational BMI, age, and symptomatology. A significantly higher percentage of placental fibrinoid was seen with women with low CTs (<25; p < 0.01). Regarding perinatal outcomes, preeclampsia was found to be significantly associated with symptomatology but not with risk factors or CT values (p < 0.01, aOR = 14.72). Moreover, 88.9% of women diagnosed with COVID-19 at <35 gestational weeks and symptomatic developed preeclampsia. (4) The data support strong guidance for pregnancies with SARS-CoV-2 infection, in particular preeclampsia and placental pathology, which need further investigation.

Prevalence, clinical features, and outcomes of SARS-CoV-2 infection in pregnant women with or without mild/moderate symptoms: Results from universal screening in a tertiary care center in Mexico City, Mexico.

The perinatal consequences of SARS-CoV-2 infection are still largely unknown. This study aimed to describe the features and outcomes of pregnant women with or without SARS-CoV-2 infection after the universal screening was established in a large tertiary care center admitting only obstetric related conditions without severe COVID-19 in Mexico City. This retrospective case-control study integrates data between April 22 and May 25, 2020, during active community transmission in Mexico, with one of the highest COVID-19 test positivity percentages worldwide. Only pregnant women and neonates with a SARS-CoV-2 result by quantitative RT-PCR were included in this study. Among 240 pregnant women, the prevalence of COVID-19 was 29% (95% CI, 24% to 35%); 86% of the patients were asymptomatic (95% CI, 76%-92%), nine women presented mild symptoms, and one patient moderate disease. No pregnancy baseline features or risk factors associated with severity of infection, including maternal age > 35 years, Body Mass Index >30 kg/m2, and pre-existing diseases, differed between positive and negative women. The median gestational age at admission for both groups was 38 weeks. All women were discharged at home without complications, and no maternal death was reported. The proportion of preeclampsia was higher in positive women than negative women (18%, 95% CI, 10%-29% vs. 9%, 95% CI, 5%-14%, P<0.05). No differences were found for other perinatal outcomes. SARS-CoV-2 test result was positive for nine infants of positive mothers detected within 24h of birth. An increased number of infected neonates were admitted to the NICU, compared to negative neonates (44% vs. 22%, P<0.05) and had a longer length of hospitalization (2 [2-18] days vs. 2 [2-3] days, P<0.001); these are potential proxies for illness severity. This report highlights the importance of COVID-19 detection at delivery in pregnant women living in high transmission areas.

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping.

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

Expression of Membrane Progesterone Receptors in Eutopic and Ectopic Endometrium of Women with Endometriosis.

Endometriosis is one of the most frequent gynecological diseases in reproductive age women, but its etiology is not completely understood. Endometriosis is characterized by progesterone resistance, which has been explained in part by a decrease in the expression of the intracellular progesterone receptor in the ectopic endometrium. Progesterone action is also mediated by nongenomic mechanisms via membrane progesterone receptors (mPRs) that belong to the class II members of the progesterone and adipoQ receptor (PAQR) family. The aim of the present study was to evaluate the expression at mRNA and protein levels of mPR members in the eutopic and ectopic endometrium of women with endometriosis. Total RNA and total protein were isolated from control endometrium (17 samples), eutopic endometrium (17 samples), and ectopic endometrium (9 samples). The expression of PAQR7 (mPRα), PAQR8 (mPRß), and PAQR6 (mPRδ) at mRNA and protein levels was evaluated by RT-qPCR and Western blot, whereas PAQR5 (mPRγ) gene expression was evaluated by RT-qPCR. Statistical analysis between comparable groups was performed using one-way ANOVA followed by Tukey's multiple comparisons test with a confidence interval of 95 %. The analysis of gene expression showed that PAQR7 and PAQR5 expression was lower in both eutopic and ectopic endometrium as compared to the endometrium of women without endometriosis, whereas the expression of PAQR8 and PAQR6 was only reduced in eutopic endometrium. Furthermore, mPRα and mPRß protein content was decreased in the ectopic endometrium of women with endometriosis. Our results demonstrate a decrease in the expression and protein content of mPRs in eutopic and ectopic endometrium of patients with endometriosis, which could contribute to the progesterone resistance observed in patients with this disease.

Macrophage Populations in Visceral Adipose Tissue from Pregnant Women: Potential Role of Obesity in Maternal Inflammation.

Obesity is associated with inflammatory changes and accumulation and phenotype polarization of adipose tissue macrophages (ATMs). Obese pregnant women have alterations in adipose tissue composition, but a detailed description of macrophage population is not available. In this study, we characterized macrophage populations in visceral adipose tissue (VAT) from pregnant women with normal, overweight, and obese pregestational weight. Immunophenotyping of macrophages from VAT biopsies was performed by flow cytometry using CD45 and CD14 as markers of hematopoietic and monocyte linage, respectively, while HLA-DR, CD11c, CD163, and CD206 were used as pro- and anti-inflammatory markers. Adipocyte number and size were evaluated by light microscopy. The results show that pregnant women that were overweight and obese during the pregestational period had adipocyte hypertrophy. Two different macrophage populations in VAT were identified: recruited macrophages (CD45⁺CD14⁺), and a novel population lacking CD45, which was considered to be a resident macrophages subset (CD45−CD14⁺). The number of resident HLA−DRlow/− macrophages showed a negative correlation with body mass index (BMI). Both resident and recruited macrophages from obese women expressed higher CD206 levels. CD11c expression was higher in resident HLA-DR⁺ macrophages from obese women. A strong correlation between CD206 and CD11c markers and BMI was observed. Our findings show that being overweight and obese in the pregestational period is associated with adipocyte hypertrophy and specific ATMs populations in VAT.

Key Clinical Factors Predicting Adipokine and Oxidative Stress Marker Concentrations among Normal, Overweight and Obese Pregnant Women Using Artificial Neural Networks.

Maternal obesity has been related to adverse neonatal outcomes and fetal programming. Oxidative stress and adipokines are potential biomarkers in such pregnancies; thus, the measurement of these molecules has been considered critical. Therefore, we developed artificial neural network (ANN) models based on maternal weight status and clinical data to predict reliable maternal blood concentrations of these biomarkers at the end of pregnancy. Adipokines (adiponectin, leptin, and resistin), and DNA, lipid and protein oxidative markers (8-oxo-2'-deoxyguanosine, malondialdehyde and carbonylated proteins, respectively) were assessed in blood of normal weight, overweight and obese women in the third trimester of pregnancy. A Back-propagation algorithm was used to train ANN models with four input variables (age, pre-gestational body mass index (p-BMI), weight status and gestational age). ANN models were able to accurately predict all biomarkers with regression coefficients greater than R² = 0.945. P-BMI was the most significant variable for estimating adiponectin and carbonylated proteins concentrations (37%), while gestational age was the most relevant variable to predict resistin and malondialdehyde (34%). Age, gestational age and p-BMI had the same significance for leptin values. Finally, for 8-oxo-2'-deoxyguanosine prediction, the most significant variable was age (37%). These models become relevant to improve clinical and nutrition interventions in prenatal care.

Epigenetic modifications in cell lines of human astrocytoma differentially regulate expression of apoptotic genes.

OBJECTS: Epigenetic alterations, known as epimutations, act by deregulating gene expression. These epimutations are reversible through the action of chromatin modifiers such as DNA methylation (DNA-met) and histone deacetylases (HDAC) inhibitors. The present study evaluated the effect of 5-azacitidine (5-aza) and sodium butyrate (NaBu) as inhibitors of DNA-met and HDAC, respectively, in the expression of genes involved in apoptosis. METHODS: D54-MG, U373-MG, and T98G cell lines were exposed to 8 mM of NaBu and 12 µM of 5-aza, as well as a combination of both, for 24 h. The expression of the Bcl-2, Bak-1, Bax, Caspase-3, and Caspase-9 genes was assessed by RT-PCR. RESULTS: They show that the Bcl-2, Caspase-3, and Caspase-9 genes were not expressed by the U373-MG and T98G lines, and that the D54-MG line did not express Bak-1. After treatment, however, these cell lines expressed all of the genes due to the effect of 5-aza on Bak-1 in D54-MG and Caspase-9 in T98G, which suggests repression by DNA-met. Meanwhile, Bcl-2, Caspase-3, and Caspase-9 were in the U373-MG and T98G lines expressed after NaBu treatment. The effect of 5-aza induced an increase in the expression of Bax and Bcl-2, while NaBu produced a similar effect on the Bak-1 and Bax genes. CONCLUSIONS: Results reveal that histone deacetylation is the principle mechanism for repressing these genes and that their basal expression is regulated primarily by this form of histone modification.

Expression of histone acetylases p300 and PCAF in pediatric astrocytomas.

OBJECTS: The protein 300 (p300) and p300/CBP-binding protein-associated factor (PCAF) are enzymes with histone acetyltransferase (HAT) activity, a function that can become deregulated in different tumors and affect biological responses. METHODS: Due to the lack of information on the deregulation of these HATs in pediatric tumors, this study evaluated the expression of both the mRNA and proteins of p300 and PCAF in 54 samples of pediatric astrocytomas embedded in paraffin. RESULTS: PCAF was not expressed in normal brain tissue. In grade I tumors, the expression of p300 (1.1 ± 0.1) and PCAF (1.2 ± 0.11) was greater than those observed in grade III tumors: 0.72 ± 0.15 for p300 and 0.55 ± 0.11 for PCAF, and grade IV tumors: 0.74 ± 0.13 for p300 and 0.55 ± 0.13 for PCAF (p < 0.05). Immunohistochemical staining revealed the same tendency towards a decrease in the expression of the protein as the degree of clinical severity increased. Patients with recurrent grades I, III, and IV tumors had the highest levels of PCAF, compared to those who showed no recurrence (p < 0.05). CONCLUSIONS: This work describes and confirms that these HATs play important roles in regulating genes and in the biological behavior of pediatric astrocytomas.

Sodium butyrate increases the effect of the photodynamic therapy: a mechanism that involves modulation of gene expression and differentiation in astrocytoma cells.

OBJECTIVES: In order to evaluate the improvement of the photodynamic therapy (PDT) due to sodium butyrate (NaBu), its effectiveness in U373-MG and D54-MG astrocytoma cell lines was evaluated. METHODS: Cells were exposed to delta-aminolevulinic acid (δ-ALA) as a precursor to endogenous photosensitizer protoporphyrin IX (PpIX). In both astrocytoma cells, an important increase by ALA was observed in uroporphyrinogen synthetase gene expression: 1.8- and 52-fold for D54-MG and U373-MG cells, respectively. After irradiation, they showed 16.67 and 28.9% of mortality in U373-MG and D54-MG, respectively. These mortalities increased to 70.62 and 96.7% when U373-MG and D54-MG cells, respectively, were exposed 24 h to 8 mM NaBu, before to PpIX induction. NaBu induced expression of caspase-3, caspase-9, and Bcl-2 and increased Bax in U373-MG cells. ALA-induced morphological changes are compatible to differentiation. CONCLUSIONS: Genes and differentiation induced mainly by NaBu improve cell death performed by PDT in astrocytoma cells. These facts prove the synergistic effect of NaBu on cytotoxic damage induced by PDT.

Effects of sodium butyrate on cell death induced by photodynamic therapy in U373-MG and D54-MG astrocytoma cell lines.

The damage induced by end products of photodynamic therapy (PDT) in astrocytoma tumors leads to cytotoxicity and cell death. Chromatin modifiers such as sodium butyrate (NaB) induce several genes involved in apoptosis, among others. The PDT improvement was evaluated by the measurement of its effectiveness in the treatment of U373-MG and D54-MG astrocytoma cell lines exposed to NaB. Cells exposed to 80 microg mL(-1) of delta-aminolevulinic acid (ALA) as precursor of endogenous photosensitizer (PS), protoporphyrin IX (PpIX), induced 16.67% and 28.9% of mortality in U373-MG and D54-MG, respectively. The mortality increased to 70.62% and 96.7%, respectively, when U373-MG and D54-MG cells were exposed for 24 h to 8 mm NaB prior to ALA-induction. In this condition, re-expression of some genes related to apoptosis in U373-MG, and differentiation in D54-MG were induced. PpIX accumulation was higher than ALA-induction and the acetylation of histone H4 induced by NaB was verified by immunocytochemistry in both cells. It can be concluded that modified chromatin and genes induced by NaB increment the cellular death induced by PDT in astrocytoma cells using PpIX as endogenous PS.