Pharmacogenomics of 17-alpha hydroxyprogesterone caproate for recurrent preterm birth: a case-control study.

OBJECTIVE: To compare maternal genotypes between women with and without significant prolongation of pregnancy in the setting of 17-alpha hydroxyprogesterone caproate (17-P) administration for the prevention of recurrent preterm birth (PTB). DESIGN: Case-control. SETTING: Three tertiary-care centres across the USA. POPULATION: Women (n = 99) with ≥ 1 prior singleton spontaneous PTB, receiving 17-P. METHODS: Women were classified as having successful prolongation of pregnancy during the 17-P treated pregnancy, in two ways: (1) Definition A: success/non-success based on difference in gestational age at delivery between 17-P-treated and untreated pregnancies (success: delivered ≥ 3 weeks later with 17-P) and (2) Definition B: success/non-success based on reaching term (success: delivered at term with 17-P). MAIN OUTCOME MEASURES: To assess genetic variation, all women underwent whole exome sequencing. Between-group sequence variation was analysed with the Variant Annotation, Analysis, and Search Tool (VAAST). Genes scored by VAAST with P < 0.05 were then analysed with two online tools: (1) Protein ANalysis THrough Evolutionary Relationships (PANTHER) and (2) Database for Annotation, Visualization, and Integrated Discovery (DAVID). RESULTS: Using Definition A, there were 70 women with successful prolongation and 29 without; 1375 genes scored by VAAST had P < 0.05. Using Definition B, 47 women had successful prolongation and 52 did not; 1039 genes scored by VAAST had P < 0.05. PANTHER revealed key differences in gene ontology pathways. Many genes from definition A were classified as prematurity genes (P = 0.026), and those from definition B as pharmacogenetic genes (P = 0.0018); (P, non-significant after Bonferroni correction). CONCLUSION: A novel analytic approach revealed several genetic differences among women delivering early vs later with 17-P. TWEETABLE ABSTRACT: Several key genetic differences are present in women with recurrent preterm birth despite 17-P treatment.

Loss of inherited genomic imprints in mice leads to severe disruption in placental lipid metabolism.

INTRODUCTION: Monoallelic expression of imprinted genes is necessary for placental development and normal fetal growth. Differentially methylated domains (DMDs) largely determine the parental-specific monoallelic expression of imprinted genes. Maternally derived DNA (cytosine-5-) -methyltransferase 1o (DNMT1o) maintains DMDs during the eight-cell stage of development. DNMT1o-deficient mouse placentas have a generalized disruption of genomic imprints. Previous studies have demonstrated that DNMT1o deficiency alters placental morphology and broadens the embryonic weight distribution in late gestation. Lipids are critical for fetal growth. Thus, we assessed the impact of disrupted imprinting on placental lipids. METHODS: Lipids were quantified from DNMT1o-deficient mouse placentas and embryos at E17.5 using a modified Folch method. Expression of select genes critical for lipid metabolism was quantified with RT-qPCR. Mitochondrial morphology was assessed by TEM and mitochondrial aconitase and cytoplasmic citrate concentrations quantified. DMD methylation was determined by EpiTYPER. RESULTS: We found that DNMT1o deficiency is associated with increased placental triacylglycerol levels. Neither fetal triacylglycerol concentrations nor expression of select genes that mediate placental lipid transport were different from wild type. Placental triacylglycerol accumulation was associated with impaired beta-oxidation and abnormal citrate metabolism with decreased mitochondrial aconitase activity and increased cytoplasmic citrate concentrations. Loss of methylation at the MEST DMD was strongly associated with placental triacylglycerol accumulation. DISCUSSION: A generalized disruption of genomic imprints leads to triacylglycerol accumulation and abnormal mitochondrial function. This could stem directly from a loss of methylation at a given DMD, such as MEST, or represent a consequence of abnormal placental development.

The influence of ligand-activated LXR on primary human trophoblasts.

INTRODUCTION: The Liver X Receptors (LXRs) are critical transcriptional regulators of cellular metabolism that promote cholesterol efflux and lipogenesis in response to excess intracellular cholesterol. In contrast, the Sterol Response Element Binding Protein-2 (SREBP2) promotes the synthesis and uptake of cholesterol. Oxysterols are products of cholesterol oxidation that accumulate in conditions associated with increased cellular levels of reactive oxygen species, such as hypoxia and oxidative stress, activating LXR and inhibiting SREBP2. While hypoxia and oxidative stress are commonly implicated in placental injury, the impact of the transcriptional regulation of cholesterol homeostasis on placental function is not well characterized. METHODS: We measured the effects of the synthetic LXR ligand T0901317 and the endogenous oxysterol 25-hydroxycholesterol (25OHC) on differentiation, cytotoxicity, progesterone synthesis, lipid droplet formation, and gene expression in primary human trophoblasts. RESULTS: Exposure to T0901317 promoted lipid droplet formation and inhibited differentiation, while 25OHC induced trophoblast toxicity, promoted hCG and progesterone release at lower concentrations with inhibition at higher concentrations, and had no effect on lipid droplet formation. The discrepant effect of these ligands was associated with distinct changes in expression of LXR and SREBP2 target genes, with upregulation of ABCA1 following 25OHC and T090317 exposure, exclusive activation of the lipogenic LXR targets SREBP1c, ACC1 and FAS by T0901317, and exclusive inhibition of the SREBP2 targets LDLR and HMGCR by 25OHC. CONCLUSION: These findings implicate cholesterol oxidation as a determinant of trophoblast function and activity, and suggest that placental gene targets and functional pathways are selectively regulated by specific LXR ligands.

Fatty acid binding protein-4 is expressed in the mouse placental labyrinth, yet is dispensable for placental triglyceride accumulation and fetal growth.

INTRODUCTION: Fatty Acid Binding Protein-4 (FABP4) is a member of a family of FABP proteins that regulate intracellular lipid trafficking in diverse tissues. We recently showed that FABP4 regulates triglyceride accumulation in primary human trophoblasts. To assess the function of placental FABP4 in vivo, we tested the hypothesis that FABP4 is expressed in the murine placenta, and regulates placenta triglyceride accumulation. METHODS: C57Bl/6 wild type or Fabp4-null mice were time-bred, and fetuses and placentas harvested at different time points during pregnancy. Placental FABP4 expression was assessed at different gestational ages, using quantitative PCR, immunohistochemistry, immunofluorescence and western immunoblotting. FABPs expression was examined by RT-qPCR. Placental lipids were extracted using the Folch method and triglyceride levels determined using a colorimetric quantification kit. RESULTS: Using immunohistochemistry, we found that FABP4 was expressed in the placental labyrinthine layer, predominantly in endothelial cells in association with CD31 positive fetal capillaries. The level of placental FABP4 mRNA and protein increased from E12.5 to E16.5 and slightly decreased at E18.5. Breeding of Fabp4 heterozygous mice resulted in embryonic genotypes that followed a Mendelian distribution and exhibited normal weight and morphology, triglyceride content, and expression of other FABP family members. Exposure to hypoxia (O2 = 12%) between E12.5-E18.5 did not uncover a difference between wild type and Fabp4-null mice. CONCLUSIONS: FABP4 is expressed in the mouse placental labyrinth, with highest expression at E16.5. FABP4 is dispensable for feto-placental growth and placental lipid accumulation.

The impact of ionizing radiation on placental trophoblasts.

INTRODUCTION: Exposure to low-dose radiation is widespread and attributable to natural sources. However, occupational, medical, accidental, and terrorist-related exposures remain a significant threat. Information on radiation injury to the feto-placental unit is scant and largely observational. We hypothesized that radiation causes trophoblast injury, and alters the expression of injury-related transcripts in vitro or in vivo, thus affecting fetal growth. METHODS: Primary human trophoblasts (PHTs), BeWo or NCCIT cells were irradiated in vitro, and cell number and viability were determined. Pregnant C57Bl/6HNsd mice were externally irradiated on E13.5, and placentas examined on E17.5. RNA expression was analyzed using microarrays and RT-qPCR. The experiments were repeated in the presence of the gramicidin S (GS)-derived nitroxide JP4-039, used to mitigate radiation-induced cell injury. RESULTS: We found that survival of in vitro-irradiated PHT cell was better than that of irradiated BeWo trophoblast cell line or the radiosensitive NCCIT mixed germ cell tumor line. Radiation altered the expression of several trophoblast genes, with a most dramatic effect on CDKN1A (p21, CIP1). Mice exposed to radiation at E13.5 exhibited a 25% reduction in mean weight by E17.5, and a 9% reduction in placental weight, which was associated with relatively small changes in placental gene expression. JP4-039 had a minimal effect on feto-placental growth or on gene expression in irradiated PHT cells or mouse placenta. DISCUSSION AND CONCLUSION: While radiation affects placental trophoblasts, the established placenta is fairly resistant to radiation, and changes in this tissue may not fully account for fetal growth restriction induced by ionizing radiation.

IFPA Meeting 2013 Workshop Report II: use of 'omics' in understanding placental development, bioinformatics tools for gene expression analysis, planning and coordination of a placenta research network, placental imaging, evolutionary approaches to understanding pre-eclampsia.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At the IFPA meeting 2013 twelve themed workshops were presented, five of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of new technologies for placenta research: 1) use of 'omics' in understanding placental development and pathologies; 2) bioinformatics and use of omics technologies; 3) planning and coordination of a placenta research network; 4) clinical imaging and pathological outcomes; 5) placental evolution.

Optimising sample collection for placental research.

Biobanks provide an important repository of samples for research purposes. However, for those samples to reflect the in vivo state, and for experimental reliability and reproducibility, careful attention to collection, processing and storage is essential. This is particularly true for the placenta, which is potentially subjected to stressful conditions during delivery, and sample collection may be delayed owing to routine postpartum inspection by clinical staff. In addition, standardisation of the collection procedure enables samples to be shared among research groups, allowing larger datasets to be established. Here, we provide an evidence-based and experts' review of the factors surrounding collection that may influence data obtained from the human placenta. We outline particular requirements for specific techniques, and propose a protocol for optimal sample collection. We recognise that the relevance of these factors, and of the sample types collected to a particular study will depend on the research questions being addressed. We therefore anticipate that researchers will select from the protocol to meet their needs and resources available. Wherever possible, we encourage researchers to extend their collection to include additional samples that can be shared on an international collaborative basis, with appropriate informed consent, to raise the quality, as well as quantity, of placental research.

Review: placenta-specific microRNAs in exosomes - good things come in nano-packages.

MicroRNAs (miRNAs) are small noncoding RNA gene products that commonly regulate mRNA expression by repression of translation and/or transcript decay. Whereas common and unique types of miRNAs are expressed by the placenta during pregnancy, the functions of most placental miRNA species are unknown. In addition to their intracellular silencing function, miRNAs are also released to the extracellular space and systemic circulation, where they can potentially target cells to regulate mRNA and protein expression, providing a non-hormonal means of intercellular communication that contributes to tissue homeostasis and disease pathophysiology. This review centers on extracellular miRNAs that originate in trophoblasts and that could mediate crosstalk between the feto-placental unit and the mother during pregnancy. We specifically detail the function of miRNAs from the primate-specific chromosome 19 miRNA cluster. These miRNAs are highly expressed in human placentas and in the serum of pregnant women. They are also packaged into extracellular vesicles of diverse sizes, including exosomes, and endow non-trophoblastic cells with resistance to a variety of viruses.

Gene targeting in primary human trophoblasts.

Studies in primary human trophoblasts provide critical insights into placental function in normal and complicated pregnancies. Mechanistic studies in these cells require experimental tools to modulate gene expression. Lipid-based methods to transfect primary trophoblasts are fairly simple to use and allow for the efficient delivery of nucleic acids, but potential toxic effects limit these methods. Viral vectors are versatile transfection tools of native trophoblastic or foreign cDNAs, providing high transfection efficiency, low toxicity and stable DNA integration into the trophoblast genome. RNA interference (RNAi), using small interfering RNA (siRNA) or microRNA, constitutes a powerful approach to silence trophoblast genes. However, off-target effects, such as regulation of unintended complementary transcripts, inflammatory responses and saturation of the endogenous RNAi machinery, are significant concerns. Strategies to minimize off-target effects include using multiple individual siRNAs, elimination of pro-inflammatory sequences in the siRNA construct and chemical modification of a nucleotide in the guide strand or of the ribose moiety. Tools for efficient gene targeting in primary human trophoblasts are currently available, albeit not yet extensively validated. These methods are critical for exploring the function of human trophoblast genes and may provide a foundation for the future application of gene therapy that targets placental trophoblasts.

Intrauterine temperature during intrapartum amnioinfusion: a prospective observational study.

OBJECTIVE: To determine the influence of routine intrapartum amnioinfusion (AI) on intrauterine temperature. DESIGN: Prospective observational study. SETTING: Maternity unit, Barnes Jewish Hospital, St Louis, MO, USA. SAMPLE: Forty women with singleton gestations and an indication for intrapartum intrauterine pressure catheter placement. METHODS: Using a temperature probe, we digitally recorded intrauterine temperature every 10 minutes during labour. Amnioinfusion was administered according to a standard protocol using saline equilibrated to the ambient temperature. MAIN OUTCOME MEASURE: Mean intrauterine temperature during labour. RESULTS: Participants were monitored for a mean of 280 minutes (range 20-820). A total of 164 intrauterine temperature readings in the AI cohort were compared with 797 control measurements. When compared with controls, we observed a lower intrauterine temperature in the AI cohort (36.4 versus 37.4°C, P<0.01). More measurements in the AI cohort were recorded in the presence of intrapartum fever (40% versus 30%). A subgroup analysis of measurements recorded in afebrile parturients revealed an even greater effect of AI (1.5°C decrease, 37.3 versus 35.8°C, P<0.01). CONCLUSION: Routine intrapartum AI using saline equilibrated to a mean ambient temperature of 25.0°C reduces intrauterine temperature and may thereby affect fetal core temperature.

IFPA Meeting 2011 workshop report I: Placenta: Predicting future health; roles of lipids in the growth and development of feto-placental unit; placental nutrient sensing; placental research to solve clinical problems--a translational approach.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2011 there were twelve themed workshops, four of which are summarized in this report. These workshops related to both basic science and clinical research into placental growth and nutrient sensing and were divided into 1) placenta: predicting future health; 2) roles of lipids in the growth and development of feto-placental unit; 3) placental nutrient sensing; 4) placental research to solve clinical problems: a translational approach.

IFPA Meeting 2011 workshop report III: Placental immunology; epigenetic and microRNA-dependent gene regulation; comparative placentation; trophoblast differentiation; stem cells.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2011 there were twelve themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology: 1) immunology; 2) epigenetics; 3) comparative placentation; 4) trophoblast differentiation; 5) stem cells.

The timing and duration of hypoxia determine gene expression patterns in cultured human trophoblasts.

OBJECTIVE: Exposure of cultured trophoblasts to hypoxia is commonly used to interrogate the molecular mechanisms underlying placental hypoxic injury. We examined the effect of levels, durations, and patterns of hypoxia on gene expression patterns in primary human trophoblasts. STUDY DESIGN: We exposed primary term human trophoblasts to either standard culture conditions (O(2) = 20%) or to static or alternating levels of oxygen (O(2) = 8%, or O(2) = 0%) either early or late in culture, and analyzed the expression of 34 genes that are known to be regulated in placentas from pregnancies complicated by fetal growth restriction (FGR). RESULTS: Using multidimensional scale analysis, Euclidean distance, and hierarchical clustering, we found that gene expression patterns in cells exposed to O(2) = 8% were similar to patterns observed in O(2) = 20%, but more distant from patterns in O(2) = 0%. Alternating atmospheric oxygen (8% vs. 0%) yielded intermediate results. Changes in oxygen levels over a longer period had a greater effect on gene expression than short-term changes. Gene expression patterns in cultured trophoblasts did not fully capture expression patterns observed in biopsies from FGR placentas vs. control. CONCLUSIONS: The level, duration, and patterns of hypoxia are critical in determining trophoblast gene expression, and therefore germane for analysis of trophoblast hypoxic injury.

The levels of hypoxia-regulated microRNAs in plasma of pregnant women with fetal growth restriction.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression at the post-transcriptional level. While mostly intracellular, a portion of cellular miRNAs is released to the circulation and their level in the plasma is altered in certain pathological conditions such as cancer, and also during pregnancy. We examined the circulating levels of a set of trophoblastic miRNAs, which we recently found to be regulated by hypoxia, in the plasma of pregnant women with fetal growth restriction (FGR). Pregnancy was associated with increased plasma levels of several placenta-specific miRNAs, compared to non-pregnant controls. Among pregnant women, the overall levels of miRNA species that we analyzed were increased by 1.84-fold (p < or = 0.01) in plasma of women with pregnancies complicated by FGR, but decreased in FGR placentas by 24% (p < or = 0.01) compared to values from uncomplicated pregnancies. Together, our results show that plasma concentration of miRNAs is regulated in pregnancy, and that FGR is associated with increased circulating miRNA levels, highlighting the need to explore plasma miRNAs as potential biomarkers for placental diseases.

The expression of connective tissue growth factor in pregnancies complicated by severe preeclampsia or fetal growth restriction.

We tested the hypothesis that the expression of placental connective tissue growth factor (CTGF) is enhanced in pregnancies complicated by severe preeclampsia (PE) or fetal growth restriction (FGR). CTGF expression was analyzed using immunostaining, western blot and real-time quantitative PCR in placental samples obtained after third trimester cesarean deliveries without labor from women with severe PE (n=11), idiopathic FGR (n=14), or healthy controls (n=14). Serum CTGF concentrations were analyzed using ELISA. We found that CTGF was stably expressed in villous trophoblasts throughout pregnancy. The expression of CTGF mRNA in placentas from severe PE or FGR was higher than placentas from controls. Whereas the levels of placental CTGF protein were similar between normal and severe PE, maternal and fetal serum CTGF levels were elevated in severe PE. Maternal CTGF levels were also distinctively elevated in women with PE or FGR with histological evidence of placental injury. The enhancement of CTGF expression as well as serum CTGF levels in clinical conditions attributed to placental dysfunction suggests a role for this secretary protein in the pathophysiology of placental injury or its sequelae.

The C5b-9 membrane attack complex of complement activation localizes to villous trophoblast injury in vivo and modulates human trophoblast function in vitro.

The complement system plays an important role in normal human pregnancy. Uncontrolled activation of this system has been associated with many disease states. We tested the hypothesis that the C5b-9 membrane attack complex (MAC) localizes to sites of villous injury and modulates trophoblast function. Placental sections from pregnancies with no complications, intrauterine growth restriction, or preeclampsia were immunostained and the surface density for MAC and fibrin was determined by morphometric analysis. Primary cytotrophoblasts from term placentas were cultured in a FiO(2) of <1%, 8% and 20% with 10% human serum containing active MAC or heat-inactivated control serum. Immunofluorescent MAC binding to trophoblast was quantified, and the neoepitopes formed in cytokeratin 18 filaments and poly-ADP-ribose polymerase during apoptosis were used to measure cell death. Trophoblast differentiation was assessed by HCG secretion, formation of syncytia, and expression of syncytin. MAC localized to fibrin deposits in normal placentas, and especially in placentas from IUGR and preeclampsia. MAC binding to cytotrophoblasts was inversely proportional to FiO(2) and enhanced apoptosis. MAC increased markers of differentiation in cultures at 72h (medium HCG, syncytia and syncytin expression). Our findings demonstrate that MAC associates with fibrin deposits at sites of villous injury in vivo. Hypoxia also enhances MAC deposition in cultured trophoblasts and MAC alters trophoblast function in a phenotype specific manner.

Hypoxia enhances the expression of follistatin-like 3 in term human trophoblasts.

Hypoxic injury hinders placental differentiation and alters trophoblast gene expression. We tested the hypothesis that the expression of follistatin-like 3 (FSTL3), a member of the follistatin family of proteins, is modulated by hypoxia in primary human trophoblast (PHT). Using immunofluorescence of human term placental villi we detected the expression of FSTL3 protein in placental villi, primarily in trophoblasts. We verified this finding in cultured term PHT cells. Basal expression of FSTL3 transcript in cultured PHT cells, determined using quantitative PCR, was stable over the culture period. Importantly, when compared to culture in FiO(2)=20% or FiO(2)=8%, PHT cells cultured in FiO(2) <1% exhibited a 4-6 fold increase in FSTL3 mRNA expression as early as 4h in hypoxia. Whereas cellular FSTL3 protein was unchanged in hypoxia, we found that hypoxia increased the level of FSTL3 in the medium. Lastly, the exposure of PHT cells to either the hypoxia-mimetic cobalt chloride or the proline hydroxylase inhibitor dimethyloxaloylglycine upregulated the expression of FSTL3 transcript. Our data indicate that hypoxia enhances the expression of FSTL3 and its release from PHT cells. Our finding that hypoxia-mimetic agents enhance FSTL3 expression implicates HIF1alpha in this process.