Reduced fetal growth velocity precedes antepartum fetal death.

OBJECTIVES: To determine whether decreased fetal growth velocity precedes antepartum fetal death and to evaluate whether fetal growth velocity is a better predictor of antepartum fetal death compared to a single fetal biometric measurement at the last available ultrasound scan prior to diagnosis of demise. METHODS: This was a retrospective, longitudinal study of 4285 singleton pregnancies in African-American women who underwent at least two fetal ultrasound examinations between 14 and 32 weeks of gestation and delivered a liveborn neonate (controls; n = 4262) or experienced antepartum fetal death (cases; n = 23). Fetal death was defined as death diagnosed at ≥ 20 weeks of gestation and confirmed by ultrasound examination. Exclusion criteria included congenital anomaly, birth at < 20 weeks of gestation, multiple gestation and intrapartum fetal death. The ultrasound examination performed at the time of fetal demise was not included in the analysis. Percentiles for estimated fetal weight (EFW) and individual biometric parameters were determined according to the Hadlock and Perinatology Research Branch/Eunice Kennedy Shriver National Institute of Child Health and Human Development (PRB/NICHD) fetal growth standards. Fetal growth velocity was defined as the slope of the regression line of the measurement percentiles as a function of gestational age based on two or more measurements in each pregnancy. RESULTS: Cases had significantly lower growth velocities of EFW (P < 0.001) and of fetal head circumference, biparietal diameter, abdominal circumference and femur length (all P < 0.05) compared to controls, according to the PRB/NICHD and Hadlock growth standards. Fetuses with EFW growth velocity < 10th percentile of the controls had a 9.4-fold and an 11.2-fold increased risk of antepartum death, based on the Hadlock and customized PRB/NICHD standards, respectively. At a 10% false-positive rate, the sensitivity of EFW growth velocity for predicting antepartum fetal death was 56.5%, compared to 26.1% for a single EFW percentile evaluation at the last available ultrasound examination, according to the customized PRB/NICHD standard. CONCLUSIONS: Given that 74% of antepartum fetal death cases were not diagnosed as small-for-gestational age (EFW < 10th percentile) at the last ultrasound examination when the fetuses were alive, alternative approaches are needed to improve detection of fetuses at risk of fetal death. Longitudinal sonographic evaluation to determine growth velocity doubles the sensitivity for prediction of antepartum fetal death compared to a single EFW measurement at the last available ultrasound examination, yet the performance is still suboptimal. © 2020 International Society of Ultrasound in Obstetrics and Gynecology.

Prediction of adverse perinatal outcome by fetal biometry: comparison of customized and population-based standards.

OBJECTIVE: To compare the predictive performance of estimated fetal weight (EFW) percentiles, according to eight growth standards, to detect fetuses at risk for adverse perinatal outcome. METHODS: This was a retrospective cohort study of 3437 African-American women. Population-based (Hadlock, INTERGROWTH-21st , World Health Organization (WHO), Fetal Medicine Foundation (FMF)), ethnicity-specific (Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)), customized (Gestation-Related Optimal Weight (GROW)) and African-American customized (Perinatology Research Branch (PRB)/NICHD) growth standards were used to calculate EFW percentiles from the last available scan prior to delivery. Prediction performance indices and relative risk (RR) were calculated for EFW < 10th and > 90th percentiles, according to each standard, for individual and composite adverse perinatal outcomes. Sensitivity at a fixed (10%) false-positive rate (FPR) and partial (FPR < 10%) and full areas under the receiver-operating-characteristics curves (AUC) were compared between the standards. RESULTS: Ten percent (341/3437) of neonates were classified as small-for-gestational age (SGA) at birth, and of these 16.4% (56/341) had at least one adverse perinatal outcome. SGA neonates had a 1.5-fold increased risk of any adverse perinatal outcome (P < 0.05). The screen-positive rate of EFW < 10th percentile varied from 6.8% (NICHD) to 24.4% (FMF). EFW < 10th percentile, according to all standards, was associated with an increased risk for each of the adverse perinatal outcomes considered (P < 0.05 for all). The highest RRs associated with EFW < 10th percentile for each adverse outcome were 5.1 (95% CI, 2.1-12.3) for perinatal mortality (WHO); 5.0 (95% CI, 3.2-7.8) for perinatal hypoglycemia (NICHD); 3.4 (95% CI, 2.4-4.7) for mechanical ventilation (NICHD); 2.9 (95% CI, 1.8-4.6) for 5-min Apgar score < 7 (GROW); 2.7 (95% CI, 2.0-3.6) for neonatal intensive care unit (NICU) admission (NICHD); and 2.5 (95% CI, 1.9-3.1) for composite adverse perinatal outcome (NICHD). Although the RR CIs overlapped among all standards for each individual outcome, the RR of composite adverse perinatal outcome in pregnancies with EFW < 10th percentile was higher according to the NICHD (2.46; 95% CI, 1.9-3.1) than the FMF (1.47; 95% CI, 1.2-1.8) standard. The sensitivity for composite adverse perinatal outcome varied substantially between standards, ranging from 15% for NICHD to 32% for FMF, due mostly to differences in FPR; this variation subsided when the FPR was set to the same value (10%). Analysis of AUC revealed significantly better performance for the prediction of perinatal mortality by the PRB/NICHD standard (AUC = 0.70) compared with the Hadlock (AUC = 0.66) and FMF (AUC = 0.64) standards. Evaluation of partial AUC (FPR < 10%) demonstrated that the INTERGROWTH-21st standard performed better than the Hadlock standard for the prediction of NICU admission and mechanical ventilation (P < 0.05 for both). Although fetuses with EFW > 90th percentile were also at risk for any adverse perinatal outcome according to the INTERGROWTH-21st (RR = 1.4; 95% CI, 1.0-1.9) and Hadlock (RR = 1.7; 95% CI, 1.1-2.6) standards, many times fewer cases (2-5-fold lower sensitivity) were detected by using EFW > 90th percentile, rather than EFW < 10th percentile, in screening by these standards. CONCLUSIONS: Fetuses with EFW < 10th percentile or EFW > 90th percentile were at increased risk of adverse perinatal outcomes according to all or some of the eight growth standards, respectively. The RR of a composite adverse perinatal outcome in pregnancies with EFW < 10th percentile was higher for the most-stringent (NICHD) compared with the least-stringent (FMF) standard. The results of the complementary analysis of AUC suggest slightly improved detection of adverse perinatal outcome by more recent population-based (INTERGROWTH-21st ) and customized (PRB/NICHD) standards compared with the Hadlock and FMF standards. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.

Evolutionary origins of the placental expression of chromosome 19 cluster galectins and their complex dysregulation in preeclampsia.

INTRODUCTION: The dysregulation of maternal-fetal immune tolerance is one of the proposed mechanisms leading to preeclampsia. Galectins are key regulator proteins of the immune response in vertebrates and maternal-fetal immune tolerance in eutherian mammals. Previously we found that three genes in a Chr19 cluster encoding for human placental galectin-13 (PP13), galectin-14 and galectin-16 emerged during primate evolution and may confer immune tolerance to the semi-allogeneic fetus. MATERIALS AND METHODS: This study involved various methodologies for gene and protein expression profiling, genomic DNA methylation analyses, functional assays on differentiating trophoblasts including gene silencing, luciferase reporter and methylation assays. These methods were applied on placental specimens, umbilical cord blood cells, primary trophoblasts and BeWo cells. Genomic DNA sequences were analyzed for transposable elements, transcription factor binding sites and evolutionary conservation. RESULTS AND DISCUSSION: The villous trophoblastic expression of Chr19 cluster galectin genes is developmentally regulated by DNA methylation and induced by key transcription factors of villous placental development during trophoblast fusion and differentiation. This latter mechanism arose via the co-option of binding sites for these transcription factors through promoter evolution and the insertion of an anthropoid-specific L1PREC2 transposable element into the 5' untranslated region of an ancestral gene followed by gene duplication events. Among placental Chr19 cluster galectin genes, the expression of LGALS13 and LGALS14 is down-regulated in preterm severe preeclampsia associated with SGA. We reveal that this phenomenon is partly originated from the dysregulated expression of key transcription factors controlling trophoblastic functions and galectin gene expression. In addition, the differential DNA methylation of these genes was also observed in preterm preeclampsia irrespective of SGA. CONCLUSIONS: These findings reveal the evolutionary origins of the placental expression of Chr19 cluster galectins. The complex dysregulation of these genes in preeclampsia may alter immune tolerance mechanisms at the maternal-fetal interface.

Enhanced tumor cures after Foscan photodynamic therapy combined with the ceramide analog LCL29. Evidence from mouse squamous cell carcinomas for sphingolipids as biomarkers of treatment response.

To improve anticancer therapeutic success of photodynamic therapy (PDT), combination treatments represent a viable strategy. Sphingolipid analogs combined with anticancer drugs can enhance tumor response. We have shown that LCL29, a C6-pyridinium ceramide, promotes therapeutic efficacy of Photofrin-PDT in mouse SCCVII squamous cell carcinoma tumors. The long-term effect of the combination PDT + LCL29 is unknown. In this study we used the same model to test the long-term curative potential of Foscan-PDT + LCL29. We show that treatment of SCCVII tumors with the combination led to enhanced long-term tumor cure compared to PDT alone. LCL29 itself did not prevent tumor growth. All treatments triggered early increases in tumor-associated C16-ceramide, C18-ceramide, dihydrosphingosine, and global levels of dihydroceramides. PDT-evoked increases in tumor-associated sphingosine-1-phosphate and dihydrosphingosine-1-phosphate remained elevated or were attenuated after the combination, respectively; in contrast, LCL29 had no effect on these two sphingolipids. Our data demonstrate that adjuvant LCL29 improves PDT long-term therapeutic efficacy, implying translational potential of the combination. Furthermore, our findings indicate that changes in the sphingolipid profile might serve as predictive biomarkers of tumor response to treatments.

Microarray profiling reveals that placental transcriptomes of early-onset HELLP syndrome and preeclampsia are similar.

BACKGROUND: The involvement of the placenta in the pathogenesis of preeclampsia and HELLP syndrome is well established, and placental lesions are also similar in these two syndromes. Here we aimed to examine the placental transcriptome and to identify candidate biomarkers in early-onset preeclampsia and HELLP syndrome. METHODS: Placental specimens were obtained at C-sections from women with early-onset preeclampsia and HELLP syndrome, and from controls who delivered preterm or at term. After histopathological examination, fresh-frozen placental specimens were used for microarray profiling and validation by qRT-PCR. Differential expression was analysed using log-linear models while adjusting for gestational age. Gene ontology and pathway analyses were used to interpret gene expression changes. Tissue microarrays were constructed from paraffin-embedded placental specimens and immunostained. RESULTS: Placental gene expression was gestational age-dependent among preterm and term controls. Out of the 350 differentially expressed genes in preeclampsia and 554 genes in HELLP syndrome, 224 genes (including LEP, CGB, LHB, INHA, SIGLEC6, PAPPA2, TREM1, and FLT1) changed in the same direction (elevated or reduced) in both syndromes. Many of these encode proteins that have been implicated as biomarkers for preeclampsia. Enrichment analyses revealed similar biological processes, cellular compartments and biological pathways enriched in early-onset preeclampsia and HELLP syndrome; however, some processes and pathways (e.g., cytokine-cytokine receptor interaction) were over-represented only in HELLP syndrome. CONCLUSION: High-throughput transcriptional and tissue microarray expression profiling revealed that placental transcriptomes of early-onset preeclampsia and HELLP syndrome largely overlap, underlying a potential common cause and pathophysiologic processes in these syndromes. However, gene expression changes may also suggest a more severe placental pathology and pronounced inflammatory response in HELLP syndrome than in preeclampsia.

Increased tumour dihydroceramide production after Photofrin-PDT alone and improved tumour response after the combination with the ceramide analogue LCL29. Evidence from mouse squamous cell carcinomas.

Photodynamic therapy (PDT) has been proven effective for treatment of several types of cancer. Photodynamic therapy alone, however, attains limited cures with some tumours and there is need for its improved efficacy in such cases. Sphingolipid (SL) analogues can promote tumour response in combination with anticancer drugs. In this study, we used mouse SCCVII squamous cell carcinoma tumours to determine the impact of Photofrin-PDT on the in vivo SL profile and the effect of LCL29, a C6-pyridinium ceramide, on PDT tumour response. Following PDT, the levels of dihydroceramides (DHceramides), in particular C20-DHceramide, were elevated in tumours. Similarly, increases in DHceramides, in addition to C20:1-ceramide, were found in PDT-treated SCCVII cells. These findings indicate the importance of the de novo ceramide pathway in Photofrin-PDT response not only in cells but also in vivo. Notably, co-exposure of SCCVII tumours to Photofrin-PDT and LCL29 led to enhanced tumour response compared with PDT alone. Thus, we show for the first time that Photofrin-PDT has a distinct signature effect on the SL profile in vitro and in vivo, and that the combined treatment advances PDT therapeutic gain, implying translational significance of the combination.

Expression patterns of microRNAs in the chorioamniotic membranes: a role for microRNAs in human pregnancy and parturition.

MicroRNAs (miRNAs) are involved in the post-transcriptional regulation of gene expression during development. This study was performed to determine gestational age-dependent changes in miRNA expression in the chorioamniotic membranes and to assess the significance of miRNAs in human pregnancy and parturition. The expression profile of 455 miRNAs was compared between patients at term without labour (TNL: n = 10), in labour (TL: n = 10), and preterm labour (PTL: n = 10) using microarrays. A total of 39 miRNAs were differentially expressed between term and preterm cases, of which 31 (79.5%) were down-regulated at term. Expression of ten miRNAs, including miR-338, differentially expressed between PTL and TL groups was decreased at term. Computational analyses using miRBase Targets have identified PLA2G4B, a phospholipase implicated in parturition, as a putative target of miR-338. Inhibition of endogenous miR-338 with anti-miR-338 increased the mRNA and protein expression of PLA2G4B in decidual cells. Luciferase assay with reporter constructs confirmed that the suppression of PLA2G4B occurs through binding of miR-338 to the 3UTR of PLA2G4B. Interestingly, the expression of Dicer, a key miRNA-processing enzyme, was markedly decreased at term, particularly with labour in the chorioamniotic membranes. Collectively, the novel findings reported herein strongly suggest that post-transcriptional regulation of genes by miRNAs, coupled with the changes of miRNA processing machinery in the chorioamniotic membranes, plays a role in pregnancy and parturition. Furthermore, the expression level of Dicer in the chorioamniotic membranes dichotomizes pathological preterm labour and physiological spontaneous labour at term.

A robust neural networks approach for spatial and intensity-dependent normalization of cDNA microarray data.

MOTIVATION: Microarray experiments are affected by numerous sources of non-biological variation that contribute systematic bias to the resulting data. In a dual-label (two-color) cDNA or long-oligonucleotide microarray, these systematic biases are often manifested as an imbalance of measured fluorescent intensities corresponding to Sample A versus those corresponding to Sample B. Systematic biases also affect between-slide comparisons. Making effective corrections for these systematic biases is a requisite for detecting the underlying biological variation between samples. Effective data normalization is therefore an essential step in the confident identification of biologically relevant differences in gene expression profiles. Several normalization methods for the correction of systemic bias have been described. While many of these methods have addressed intensity-dependent bias, few have addressed both intensity-dependent and spatiality-dependent bias. RESULTS: We present a neural network-based normalization method for correcting the intensity- and spatiality-dependent bias in cDNA microarray datasets. In this normalization method, the dependence of the log-intensity ratio (M) on the average log-intensity (A) as well as on the spatial coordinates (X,Y) of spots is approximated with a feed-forward neural network function. Resistance to outliers is provided by assigning weights to each spot based on how distant their M values is from the median over the spots whose A values are similar, as well as by using pseudospatial coordinates instead of spot row and column indices. A comparison of the robust neural network method with other published methods demonstrates its potential in reducing both intensity-dependent bias and spatial-dependent bias, which translates to more reliable identification of truly regulated genes.