Customizing EV-CATCHER to Purify Placental Extracellular Vesicles from Maternal Plasma to Detect Placental Pathologies.

Placenta Accreta Spectrum (PAS) is a life-threatening condition in which placental trophoblastic cells abnormally invade the uterus, often up to the uterine serosa and, in extreme cases, tissues beyond the uterine wall. Currently, there is no clinical assay for the non-invasive detection of PAS, and only ultrasound and MRI can be used for its diagnosis. Considering the subjectivity of visual assessment, the detection of PAS necessitates a high degree of expertise and, in some instances, can lead to its misdiagnosis. In clinical practice, up to 50% of pregnancies with PAS remain undiagnosed until delivery, and it is associated with increased risk of morbidity/mortality. Although many studies have evaluated the potential of fetal biomarkers circulating in maternal blood, very few studies have evaluated the potential of circulating placental extracellular vesicles (EVs) and their miRNA contents for molecular detection of PAS. Thus, to purify placental EVs from maternal blood, we customized our robust ultra-sensitive immuno-purification assay, termed EV-CATCHER, with a monoclonal antibody targeting the membrane Placental Alkaline Phosphatase (PLAP) protein, which is unique to the placenta and present on the surface of placental EVs. Then, as a pilot evaluation, we compared the miRNA expression profiles of placental EVs purified from the maternal plasma of women diagnosed with placenta previa (controls, n = 16); placenta lying low in uterus but not invasive) to those of placental EVs purified from the plasma of women with placenta percreta (cases, n = 16), PAS with the highest level of invasiveness. Our analyses reveal that miRNA profiling of PLAP+ EVs purified from maternal plasma identified 40 differentially expressed miRNAs when comparing these two placental pathologies. Preliminary miRNA pathway enrichment and gene ontology analysis of the top 14 upregulated and top nine downregulated miRNAs in PLAP+ EVs, purified from the plasma of women diagnosed with placenta percreta versus those diagnosed with placenta previa, suggests a potential role in control of cellular invasion and motility that will require further investigation.

Lipid profile of circulating placental extracellular vesicles during pregnancy identifies foetal growth restriction risk.

Small-for-gestational age (SGA) neonates exhibit increased perinatal morbidity and mortality, and a greater risk of developing chronic diseases in adulthood. Currently, no effective maternal blood-based screening methods for determining SGA risk are available. We used a high-resolution MS/MSALL shotgun lipidomic approach to explore the lipid profiles of small extracellular vesicles (sEV) released from the placenta into the circulation of pregnant individuals. Samples were acquired from 195 normal and 41 SGA pregnancies. Lipid profiles were determined serially across pregnancy. We identified specific lipid signatures of placental sEVs that define the trajectory of a normal pregnancy and their changes occurring in relation to maternal characteristics (parity and ethnicity) and birthweight centile. We constructed a multivariate model demonstrating that specific lipid features of circulating placental sEVs, particularly during early gestation, are highly predictive of SGA infants. Lipidomic-based biomarker development promises to improve the early detection of pregnancies at risk of developing SGA, an unmet clinical need in obstetrics.

Ancestry dependent balancing selection of placental dysferlin at high-altitude.

Introduction: The placenta mediates fetal growth by regulating gas and nutrient exchange between the mother and the fetus. The cell type in the placenta where this nutrient exchange occurs is called the syncytiotrophoblast, which is the barrier between the fetal and maternal blood. Residence at high-altitude is strongly associated with reduced 3rd trimester fetal growth and increased rates of complications such as preeclampsia. We asked whether altitude and/or ancestry-related placental gene expression contributes to differential fetal growth under high-altitude conditions, as native populations have greater fetal growth than migrants to high-altitude. Methods: We have previously shown that methylation differences largely accounted for altitude-associated differences in placental gene expression that favor improved fetal growth among high-altitude natives. We tested for differences in DNA methylation between Andean and European placental samples from Bolivia [La Paz (∼3,600 m) and Santa Cruz, Bolivia (∼400 m)]. One group of genes showing significant altitude-related differences are those involved in cell fusion and membrane repair in the syncytiotrophoblast. Dysferlin (DYSF) shows greater expression levels in high- vs. low-altitude placentas, regardless of ancestry. DYSF has a single nucleotide variant (rs10166384;G/A) located at a methylation site that can potentially stimulate or repress DYSF expression. Following up with individual DNA genotyping in an expanded sample size, we observed three classes of DNA methylation that corresponded to individual genotypes of rs10166384 (A/A < A/G < G/G). We tested whether these genotypes are under Darwinian selection pressure by sequencing a ∼2.5 kb fragment including the DYSF variants from 96 Bolivian samples and compared them to data from the 1000 genomes project. Results: We found that balancing selection (Tajima's D = 2.37) was acting on this fragment among Andeans regardless of altitude, and in Europeans at high-altitude (Tajima's D = 1.85). Discussion: This supports that balancing selection acting on dysferlin is capable of altering DNA methylation patterns based on environmental exposure to high-altitude hypoxia. This finding is analogous to balancing selection seen frequency-dependent selection, implying both alleles are advantageous in different ways depending on environmental circumstances. Preservation of the adenine (A) and guanine (G) alleles may therefore aid both Andeans and Europeans in an altitude dependent fashion.

Decreased Vitamin D Levels and Altered Placental Vitamin D Gene Expression at High Altitude: Role of Genetic Ancestry.

High-altitude hypoxia challenges reproduction; particularly in non-native populations. Although high-altitude residence is associated with vitamin D deficiency, the homeostasis and metabolism of vitamin D in natives and migrants remain unknown. We report that high altitude (3600 m residence) negatively impacted vitamin D levels, with the high-altitude Andeans having the lowest 25-OH-D levels and the high-altitude Europeans having the lowest 1α,25-(OH)2-D levels. There was a significant interaction of genetic ancestry with altitude in the ratio of 1α,25-(OH)2-D to 25-OH-D; with the ratio being significantly lower in Europeans compared to Andeans living at high altitude. Placental gene expression accounted for as much as 50% of circulating vitamin D levels, with CYP2R1 (25-hydroxylase), CYP27B1 (1α-hydroxylase), CYP24A1 (24-hydroxylase), and LRP2 (megalin) as the major determinants of vitamin D levels. High-altitude residents had a greater correlation between circulating vitamin D levels and placental gene expression than low-altitude residents. Placental 7-dehydrocholesterol reductase and vitamin D receptor were upregulated at high altitude in both genetic-ancestry groups, while megalin and 24-hydroxylase were upregulated only in Europeans. Given that vitamin D deficiency and decreased 1α,25-(OH)2-D to 25-OH-D ratios are associated with pregnancy complications, our data support a role for high-altitude-induced vitamin D dysregulation impacting reproductive outcomes, particularly in migrants.

Diminished trophoblast differentiation in early onset preeclampsia.

The mechanism by which human cytotrophoblast cells (CTB) differentiate into extravillous trophoblast cells (EVT) is an epithelial-mesenchymal transition (EMT). Polarized CTB, anchored in an epithelial layer, are transformed into motile, non-polar EVT which invade the uterus. Our previous research has shown that over gestation, invasive first trimester EVT are converted to a non-invasive phenotype showing a reduced degree of EMT. We hypothesized that in an under-invasion pathology, such as early onset preeclampsia, third trimester EVT would display a less advanced EMT profile than controls. The goal of this study was to determine whether expression of EMT-associated genes in the EVT of early onset preeclamptics shows a less mesenchymal, more epithelial phenotype compared to control pregnancies. Measures of preeclamptic CTB and EVT gene expression, using highly purified cells from third trimester, early onset preeclamptics and gestational-age matched controls, showed clear evidence of a phenotypic pattern characteristic of an EMT. Comparison of preeclamptic EVT to gestational-age matched, control EVT demonstrated multiple changes in gene expression, including changes in well-known EMT gene markers, indicative of a more limited EMT. These changes are not explained by differences in the preeclamptic CTB precursors. In this first study of purified third trimester EVT, we show that the pattern of gene expression corresponding to EMT-associated differentiation is diminished in early onset preeclampsia. This provides a mechanistic framework for many of the molecular changes observed in preeclampsia and presents an opportunity for detailed studies of the pathways regulating the aberrant EMT and for potential biomarkers of the process.

Trophoblast invasion: Lessons from abnormally invasive placenta (placenta accreta).

The invasion of the uterine wall by extravillous trophoblast is acknowledged as a crucial component of the establishment of pregnancy however, the only part of this process that has been clearly identified is the differentiation of cytotrophoblast (CTB) into the invasive extravillous trophoblast (EVT). The control of invasion, both initiation and termination, have yet to be elucidated and even the mechanism of differentiation is unclear. This review describes our studies which are designed to characterize the intracellular mechanisms that drive differentiation. We have used the over-invasion observed in abnormally invasive placenta (AIP; placenta accreta) to further interrogate this mechanism. Our results show that first trimester CTB to EVT differentiation is accomplished via an epithelial-mesenchymal transition (EMT), with EVT displaying a metastable, mesenchymal phenotype. In the third trimester, while the invasiveness of the EVT is lost, these cells still demonstrate signs of the EMT, albeit diminished. EVT isolated from AIP pregnancies do not however, show the same degree of reduction in EMT shown by normal third trimester cells. They exhibit a more mesenchymal phenotype, consistent with a legacy of greater invasiveness. The master regulatory transcription factor controlling the EMT appears, from the observational data, to be ZEB2 (zinc finger E-box binding protein 2). We verified this by overexpressing ZEB2 in the BeWo and JEG3 trophoblast cell lines and showing that they became more stellate in shape, up-regulated the expression of EMT-associated genes and demonstrated a substantially increased degree of invasiveness. The identification of the differentiation mechanism will enable us to identify the factors controlling invasion and those aberrant processes which generate the abnormal invasion seen in pathologies such as AIP and preeclampsia.

Allele-specific DNA methylation is increased in cancers and its dense mapping in normal plus neoplastic cells increases the yield of disease-associated regulatory SNPs.

BACKGROUND: Mapping of allele-specific DNA methylation (ASM) can be a post-GWAS strategy for localizing regulatory sequence polymorphisms (rSNPs). The advantages of this approach, and the mechanisms underlying ASM in normal and neoplastic cells, remain to be clarified. RESULTS: We perform whole genome methyl-seq on diverse normal cells and tissues and three cancer types. After excluding imprinting, the data pinpoint 15,112 high-confidence ASM differentially methylated regions, of which 1838 contain SNPs in strong linkage disequilibrium or coinciding with GWAS peaks. ASM frequencies are increased in cancers versus matched normal tissues, due to widespread allele-specific hypomethylation and focal allele-specific hypermethylation in poised chromatin. Cancer cells show increased allele switching at ASM loci, but disruptive SNPs in specific classes of CTCF and transcription factor binding motifs are similarly correlated with ASM in cancer and non-cancer. Rare somatic mutations affecting these same motif classes track with de novo ASM. Allele-specific transcription factor binding from ChIP-seq is enriched among ASM loci, but most ASM differentially methylated regions lack such annotations, and some are found in otherwise uninformative "chromatin deserts." CONCLUSIONS: ASM is increased in cancers but occurs by a shared mechanism involving disruptive SNPs in CTCF and transcription factor binding sites in both normal and neoplastic cells. Dense ASM mapping in normal plus cancer samples reveals candidate rSNPs that are difficult to find by other approaches. Together with GWAS data, these rSNPs can nominate specific transcriptional pathways in susceptibility to autoimmune, cardiometabolic, neuropsychiatric, and neoplastic diseases.

Human placental glucose transport in fetoplacental growth and metabolism.

While efficient glucose transport is essential for all cells, in the case of the human placenta, glucose transport requirements are two-fold; provision of glucose for the growing fetus in addition to the supply of glucose required the changing metabolic needs of the placenta itself. The rapidly evolving environment of placental cells over gestation has significant consequences for the development of glucose transport systems. The two-fold transport requirement of the placenta means also that changes in expression will have effects not only for the placenta but also for fetal growth and metabolism. This review will examine the localization, function and evolution of placental glucose transport systems as they are altered with fetal development and the transport and metabolic changes observed in pregnancy pathologies.

3D Bioprinted GelMA Based Models for the Study of Trophoblast Cell Invasion.

Bioprinting is an emerging and promising technique for fabricating 3D cell-laden constructs for various biomedical applications. In this paper, we employed 3D bioprinted GelMA-based models to investigate the trophoblast cell invasion phenomenon, enabling studies of key placental functions. Initially, a set of optimized material and process parameters including GelMA concentration, UV crosslinking time and printing configuration were identified by systematic, parametric study. Following this, a multiple-ring model (2D multi-ring model) was tested with the HTR-8/SVneo trophoblast cell line to measure cell movement under the influence of EGF (chemoattractant) gradients. In the multi-ring model, the cell front used as a cell invasion indicator moves at a rate of 85 ± 33 µm/day with an EGF gradient of 16 µM. However, the rate was dramatically reduced to 13 ± 5 µm/day, when the multi-ring model was covered with a GelMA layer to constrain cells within the 3D environment (3D multi-ring model). Due to the geometric and the functional limitations of multi-ring model, a multi-strip model (2D multi-strip model) was developed to investigate cell movement in the presence and absence of the EGF chemoattractant. The results show that in the absence of an overlying cell-free layer of GelMA, movement of the cell front shows no significant differences between control and EGF-stimulated rates, due to the combination of migration and proliferation at high cell density (6 × 106 cells/ml) near the GelMA surface. When the model was covered by a layer of GelMA (3D multi-strip model) and migration was excluded, EGF-stimulated cells showed an invasion rate of 21 ± 3 µm/day compared to the rate for unstimulated cells, of 5 ± 4 µm/day. The novel features described in this report advance the use of the 3D bioprinted placental model as a practical tool for not only measurement of trophoblast invasion but also the interaction of invading cells with other tissue elements.

Human placental GLUT1 glucose transporter expression and the fetal insulin-like growth factor axis in pregnancies complicated by diabetes.

We have previously described regulation of syncytial GLUT1 glucose transporters by IGF-I. Despite this, it is not clear what signal regulates transplacental glucose transport. In this report we asked whether changes in GLUT1 expression and glucose transport activity in diabetic pregnancies were associated with alterations in the fetal IGF axis. Cord blood samples and paired syncytial microvillous and basal membranes were isolated from normal term pregnancies and pregnancies characterized by gestational diabetes type A2 (GDM A2) and pre-existing insulin-dependent diabetes mellitus (IDDM). Circulating IGF-I, basal membrane GLUT1 expression and glucose transporter activity were correlated with birth weight, but only in control, not diabetic groups. Basal membrane GLUT1 and transporter activity were correlated with IGF-I concentrations in control, but not diabetic groups. IGF binding protein (IGFBP) binding capacity showed a ≥50% reduction in the diabetic groups compared to control; both showed a higher level of free IGF-I. The absence of a correlation between birth weight and factors such as fetal IGF-I or GLUT1 expression in the diabetic groups suggests that IGF-I-stimulated effects may have reached a limiting threshold, such that further increases in IGF-I (or GLUT1) are without effect. These data support that fetal IGF-I acts as a fetal nutritional signal, modulating placental GLUT1 expression and birth weight via altered levels of fetal circulating IGFBPs. Diabetes appears to exert its effects on fetal and placental factors prior to the third trimester and, despite good glycemic control immediately prior to, and in the third trimester, these effects persist to term.

IFPA meeting 2018 workshop report II: Abnormally invasive placenta; inflammation and infection; preeclampsia; gestational trophoblastic disease and drug delivery.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2018 there were nine themed workshops, five of which are summarised in this report. These workshops discussed new perspectives and knowledge in the following areas of research: 1) preeclampsia; 2) abnormally invasive placenta; 3) placental infection; 4) gestational trophoblastic disease; 4) drug delivery to treat placental dysfunction.

ZEB2, a master regulator of the epithelial-mesenchymal transition, mediates trophoblast differentiation.

STUDY QUESTION: Does the upregulation of the zinc finger E-box binding homeobox 2 (ZEB2) transcription factor in human trophoblast cells lead to alterations in gene expression consistent with an epithelial-mesenchymal transition (EMT) and a consequent increase in invasiveness? SUMMARY ANSWER: Overexpression of ZEB2 results in an epithelial-mesenchymal shift in gene expression accompanied by a substantial increase in the invasive capacity of human trophoblast cells. WHAT IS KNOWN ALREADY: In-vivo results have shown that cytotrophoblast differentiation into extravillous trophoblast involves an epithelial-mesenchymal transition. The only EMT master regulatory factor which shows changes consistent with extravillous trophoblast EMT status and invasive capacity is the ZEB2 transcription factor. STUDY DESIGN, SIZE, DURATION: This study is a mechanistic investigation of the role of ZEB2 in trophoblast differentiation. We generated stable ZEB2 overexpression clones using the epithelial BeWo and JEG3 choriocarcinoma lines. Using these clones, we investigated the effects of ZEB2 overexpression on the expression of EMT-associated genes and proteins, cell morphology and invasive capability. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used lentiviral transduction to overexpress ZEB2 in BeWo and JEG3 cells. Stable clones were selected based on ZEB2 expression and morphology. A PCR array of EMT-associated genes was used to probe gene expression. Protein measurements were performed by western blotting. Gain-of-function was assessed by quantitatively measuring cell invasion rates using a Transwell assay, a 3D bioprinted placenta model and the xCelligenceTM platform. MAIN RESULTS AND THE ROLE OF CHANCE: The four selected clones (2 × BeWo, 2 × JEG3, based on ZEB2 expression and morphology) all showed gene expression changes indicative of an EMT. The two clones (1 × BeWo, 1 × JEG3) showing >40-fold increase in ZEB2 expression also displayed increased ZEB2 protein; the others, with increases in ZEB2 expression <14-fold did not. The two high ZEB2-expressing clones demonstrated robust increases in invasive capacity, as assessed by three types of invasion assay. These data identify ZEB2-mediated transcription as a key mechanism transforming the epithelial-like trophoblast into cells with a mesenchymal, invasive phenotype. LARGE SCALE DATA: PCR array data have been deposited in the GEO database under accession number GSE116532. LIMITATIONS, REASONS FOR CAUTION: These are in-vitro studies using choriocarcinoma cells and so the results should be interpreted in view of these limitations. Nevertheless, the data are consistent with in-vivo findings and are replicated in two different cell lines. WIDER IMPLICATIONS OF THE FINDINGS: The combination of these data with the in-vivo findings clearly identify ZEB2-mediated EMT as the mechanism for cytotrophoblast differentiation into extravillous trophoblast. Having characterized these cellular mechanisms, it will now be possible to identify the intracellular and extracellular regulatory components which control ZEB2 and trophoblast differentiation. It will also be possible to identify the aberrant factors which alter differentiation in invasive pathologies such as preeclampsia and abnormally invasive placenta (AKA accreta, increta, percreta). STUDY FUNDING AND COMPETING INTEREST(s): Funding was provided by the Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine and Surgery at Hackensack Meridian Health, Hackensack, NJ. The 3D bioprinted placental model work done in Drs Kim and Fisher's labs was supported by the Children's National Medical Center. The xCELLigence work done in Dr Birge's lab was supported by NIH CA165077. The authors declare no competing interests.

Human trophoblast epithelial-mesenchymal transition in abnormally invasive placenta.

Differentiation of first trimester human placental cytotrophoblast (CTB) from an anchorage-dependent epithelial phenotype into the mesenchymal-like invasive extravillous trophoblast (EVT) is crucial in the development of the maternal-fetal interface. We showed previously that differentiation of first trimester CTB to EVT involves an epithelial-mesenchymal transition (EMT). Here we compare the epithelial-mesenchymal characteristics of CTB and EVT derived from normal third trimester placenta or placenta previa versus abnormally invasive placenta (AIP). CTB and EVT were isolated from normal term placenta or placenta previa following Caesarean section and EVT from AIP following Caesarean hysterectomy. Cell identity was validated by measurement of cytokeratin-7 and HLA-G. Comparing normal term CTB with EVT from normal term placenta or placenta previa for differential expression analysis of genes associated with the EMT showed changes in >70% of the genes probed. While demonstrating a mesenchymal phenotype relative to CTB, many of the gene expression changes in third trimester EVT were reduced relative to the first trimester EVT. We suggest that third trimester EVT are in a more constrained, metastable state compared to first trimester equivalents. By contrast, EVT from AIP demonstrate characteristics that are more mesenchymal than normal third trimester EVT, placing them closer to first trimester EVT on the EMT spectrum, consistent with a more invasive phenotype.

Down-regulation of the placental BCRP/ABCG2 transporter in response to hypoxia signaling.

INTRODUCTION: The BCRP/ABCG2 efflux transporter protects the developing fetus by limiting the transplacental transfer of drugs and chemicals and prevents the apoptosis of trophoblasts. The purpose of this study was to determine whether hypoxia-related signaling alters placental BCRP expression and function in vitro and in human pregnancies. METHODS: Human BeWo choriocarcinoma cells were treated with the hypoxia mimetic, cobalt chloride (CoCl2), or 3% oxygen for 24-48 h. Activation of HIF-1α signaling and regulation of BCRP was assessed using qPCR, ELISA, western blotting and a fluorescent substrate transport assay. In addition, healthy term placentas from high altitude pregnancies with chronic hypoxia were assessed for BCRP expression. RESULTS: CoCl2 and 3% oxygen increased HIF-1α protein signaling and decreased the mRNA and protein expression of BCRP by 30-75% in BeWo cells. Reduced BCRP expression corresponded with impaired efflux activity during hypoxia as evidenced by accumulation of the substrate Hoechst 33342. A number of transcription factors known to regulate BCRP, including AHR, NRF2 and PPARγ, were also coordinately down-regulated by 3% oxygen in BeWo cells. Moreover, women who gave birth at a high altitude (3100 m) exhibited signs of chronic placental hypoxia, including enhanced protein expression of the HIF-1α target GLUT1, and had reduced BCRP levels in microvillous membranes compared to women at a moderate altitude (1600 m). DISCUSSION: This study provides novel insight into the regulation of the placental BCRP transporter by hypoxia, which may be important for exposure of the fetus to chemicals during early development and in hypoxia-related pregnancy disorders.

Placental origins of adverse pregnancy outcomes: potential molecular targets: an Executive Workshop Summary of the Eunice Kennedy Shriver National Institute of Child Health and Human Development.

Although much progress is being made in understanding the molecular pathways in the placenta that are involved in the pathophysiology of pregnancy-related disorders, a significant gap exists in the utilization of this information for the development of new drug therapies to improve pregnancy outcome. On March 5-6, 2015, the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health sponsored a 2-day workshop titled Placental Origins of Adverse Pregnancy Outcomes: Potential Molecular Targets to begin to address this gap. Particular emphasis was given to the identification of important molecular pathways that could serve as drug targets and the advantages and disadvantages of targeting these particular pathways. This article is a summary of the proceedings of that workshop. A broad number of topics were covered that ranged from basic placental biology to clinical trials. This included research in the basic biology of placentation, such as trophoblast migration and spiral artery remodeling, and trophoblast sensing and response to infectious and noninfectious agents. Research findings in these areas will be critical for the formulation of the development of future treatments and the development of therapies for the prevention of a number of pregnancy disorders of placental origin that include preeclampsia, fetal growth restriction, and uterine inflammation. Research was also presented that summarized ongoing clinical efforts in the United States and in Europe that has tested novel interventions for preeclampsia and fetal growth restriction, including agents such as oral arginine supplementation, sildenafil, pravastatin, gene therapy with virally delivered vascular endothelial growth factor, and oxygen supplementation therapy. Strategies were also proposed to improve fetal growth by the enhancement of nutrient transport to the fetus by modulation of their placental transporters and the targeting of placental mitochondrial dysfunction and oxidative stress to improve placental health. The roles of microRNAs and placental-derived exosomes, as well as messenger RNAs, were also discussed in the context of their use for diagnostics and as drug targets. The workshop discussed the aspect of safety and pharmacokinetic profiles of potential existing and new therapeutics that will need to be determined, especially in the context of the unique pharmacokinetic properties of pregnancy and the hurdles and pitfalls of the translation of research findings into practice. The workshop also discussed novel methods of drug delivery and targeting during pregnancy with the use of macromolecular carriers, such as nanoparticles and biopolymers, to minimize placental drug transfer and hence fetal drug exposure. In closing, a major theme that developed from the workshop was that the scientific community must change their thinking of the pregnant woman and her fetus as a vulnerable patient population for which drug development should be avoided, but rather be thought of as a deprived population in need of more effective therapeutic interventions.

Differentiation of first trimester cytotrophoblast to extravillous trophoblast involves an epithelial-mesenchymal transition.

The transformation of cytotrophoblast (CTB) to extravillous trophoblast (EVT) is an essential process for placental implantation. EVT generated at the tips of the anchoring villi migrate away from the placenta and invade the endometrium and maternal spiral arteries, where they modulate maternal immune responses and remodel the arteries into high-volume conduits to facilitate uteroplacental blood flow. The process of EVT differentiation has several factors in common with the epithelial-to-mesenchymal transition (EMT) observed in embryonic development, wound healing and cancer metastasis. We hypothesized that the generation of invasive EVT from CTB was a form of EMT. We isolated paired CTB and EVT from first trimester placentae, and compared their gene expression using a PCR array comprising probes for genes involved in EMT. Out of 84 genes, 24 were down-regulated in EVT compared to CTB, including epithelial markers such as E-cadherin (-11-fold) and occludin (-75-fold). Another 30 genes were up-regulated in EVT compared to CTB including mesenchymal markers such as vimentin (235-fold) and fibronectin (107-fold) as well as the matrix metalloproteinases, MMP2 and MMP9 (357-fold, 129-fold). These alterations also included major increases in the ZEB2 (zinc finger E-box binding homeobox 2, 198-fold) and TCF4 (transcription factor 4, 18-fold) transcription factors, suggesting possible stimulatory mechanisms. There was substantial up-regulation of the genes encoding TGFß1 and TGFß2 (48-fold, 115-fold), which may contribute to the maintenance of the mesenchymal-like phenotype. We conclude that transformation of CTB to EVT is consistent with an EMT, although the differences with other types of EMT suggest this may be a unique form.

Three-Dimensional Power Doppler Ultrasonography for Diagnosing Abnormally Invasive Placenta and Quantifying the Risk.

OBJECTIVE: To test an objective ultrasound marker for diagnosing the presence and severity of abnormally invasive placenta. METHODS: Women at risk of abnormally invasive placenta underwent a three-dimensional power Doppler ultrasound scan. The volumes were examined offline by a blinded observer. The largest area of confluent three-dimensional power Doppler signal (Area of Confluence [Acon], cm) at the uteroplacental interface was measured and compared in women subsequently diagnosed with abnormally invasive placenta and women in a control group who did not have abnormally invasive placenta. Receiver operating characteristic curves were plotted for prediction of abnormally invasive placenta and abnormally invasive placenta requiring cesarean hysterectomy. RESULTS: Ninety-three women were recruited. Results were available for 89. Abnormally invasive placenta was clinically diagnosed in 42 women; 36 required hysterectomy and had abnormally invasive placenta confirmed histopathologically. Median and interquartile range for Acon was greater for abnormally invasive placenta (44.2 [31.4-61.7] cm) compared with women in the control group (4.5 cm [2.9-6.6], P<.001) and even greater in the 36 requiring hysterectomy (46.6 cm [37.2-72.6], P<.001). Acon rose with histopathologic diagnosis: focal accreta (32.2 cm [17.2-57.3]), accreta (59.6 cm [40.1-89.9]), and percreta (46.6 cm [37.5-71.5]; P<.001 analysis of variance for linear trend). Receiver operating characteristic analysis for prediction of abnormally invasive placenta revealed that with an Acon of 12.4 cm or greater, 100% sensitivity (95% confidence interval [CI] 91.6-100) could be obtained with 92% specificity (95% CI 79.6-97.6); area under the curve is 0.99 (95% CI 0.94-1.0). For prediction of abnormally invasive placenta requiring hysterectomy, 100% sensitivity (95% CI 90.3-100) can be obtained with an Acon of 17.4 cm or greater with 87% specificity (95% CI 74.7-94.5; area under the curve 0.98 [0.93-1.0]). CONCLUSION: The marker Acon provides a quantitative means for diagnosing abnormally invasive placenta and assessing severity. If further validated, subjectivity could be eliminated from the diagnosis of abnormally invasive placenta. LEVEL OF EVIDENCE: II.

Regulation of human trophoblast GLUT1 glucose transporter by insulin-like growth factor I (IGF-I).

Glucose transport to the fetus across the placenta takes place via glucose transporters in the opposing faces of the barrier layer, the microvillous and basal membranes of the syncytiotrophoblast. While basal membrane content of the GLUT1 glucose transporter appears to be the rate-limiting step in transplacental transport, the factors regulating transporter expression and activity are largely unknown. In view of the many studies showing an association between IGF-I and fetal growth, we investigated the effects of IGF-I on placental glucose transport and GLUT1 transporter expression. Treatment of BeWo choriocarcinoma cells with IGF-I increased cellular GLUT1 protein. There was increased basolateral (but not microvillous) uptake of glucose and increased transepithelial transport of glucose across the BeWo monolayer. Primary syncytial cells treated with IGF-I also demonstrated an increase in GLUT1 protein. Term placental explants treated with IGF-I showed an increase in syncytial basal membrane GLUT1 but microvillous membrane GLUT1 was not affected. The placental dual perfusion model was used to assess the effects of fetally perfused IGF-I on transplacental glucose transport and syncytial GLUT1 content. In control perfusions there was a decrease in transplacental glucose transport over the course of the perfusion, whereas in tissues perfused with IGF-I through the fetal circulation there was no change. Syncytial basal membranes from IGF-I perfused tissues showed an increase in GLUT1 content. These results demonstrate that IGF-I, whether acting via microvillous or basal membrane receptors, increases the basal membrane content of GLUT1 and up-regulates basal membrane transport of glucose, leading to increased transepithelial glucose transport. These observations provide a partial explanation for the mechanism by which IGF-I controls nutrient supply in the regulation of fetal growth.

Maternal and fetal outcomes in placenta accreta after institution of team-managed care.

INTRODUCTION: Placenta accreta significantly contributes to maternal morbidity and mortality. We evaluated whether planned delivery and experienced, team-managed surgical intervention results in improved outcomes. We also examined whether risk factors differed for accreta, increta, and percreta and evaluated whether excess lower segment uterine vascularity correlates with disease severity. METHODS: We retrospectively analyzed patients before versus after institution of a management protocol. Of the 58 044 deliveries over 10 years, there were 67 women whose pregnancies were histopathologically confirmed as placenta accreta, increta, or percreta (1/866). Clinical outcome measures were estimated blood loss (EBL), packed red blood cells (pRBCs) transfused, maternal and fetal complications, intensive care unit admission, and length of stay. RESULTS: There were no maternal or infant deaths. In the managed cohort, EBL was reduced by 48% (P < .001), intraoperative pRBCs transfused by 40% (P < .01), total transfused pRBCs per case by 50% (P < .01), and surgical intensive care unit admissions by >50% (P < .01). Assessment of maternal risk factors by diagnosis revealed marked differences between accreta versus increta and percreta. Clinically assessed excess vascularity of the lower uterine segment correlated with disease severity. The incidence of neonatal complications was similar in both cohorts. CONCLUSIONS: Targeted delivery at 34 weeks and team-managed diagnosis, treatment, and care of patients with placenta accreta were associated with improved maternal, but not neonatal outcomes.

Maternal and fetoplacental hypoxia do not alter circulating angiogenic growth effectors during human pregnancy.

One causal model of preeclampsia (PE) postulates that placental hypoxia alters the production of angiogenic growth effectors (AGEs), causing an imbalance leading to maternal endothelial cell dysfunction. We tested this model using the natural experiment of high-altitude (HA) residence. We hypothesized that in HA pregnancies 1) circulating soluble fms-like tyrosine kinase 1 (sFlt-1) is increased and placental growth factor (PlGF) decreased, and 2) AGE concentrations correlate with measures of hypoxia. A cross-sectional study of healthy pregnancies at low altitude (LA) (400 m) versus HA (3600 m) compared normal (n = 80 at HA, n = 90 at LA) and PE pregnancies (n = 20 PE at HA, n = 19 PE at LA). Blood was collected using standard serum separation and, in parallel, by a method designed to inhibit platelet activation. AGEs were measured by enzyme-linked immunosorbent assays. AGEs did not differ between altitudes in normal or PE pregnancies. AGE concentrations were unrelated to measures of maternal or fetal hypoxia. PlGF was lower and sFlt-1 higher in PE, but overlapped considerably with the range observed in normal samples. PlGF correlated with placental mass in both normal and PE pregnancies. The contribution of peripheral cells to the values measured for AGEs was similar at LA and HA, but was greater in PE than in normotensive women. Hypoxia, across a wide physiological range in pregnancy, does not alter levels of circulating AGEs in otherwise normal pregnancies. Peripheral cell release of AGEs with the hemostasis characteristic of standard blood collection is highly variable and contributes to a doubling of the amount of sFlt-1 measured in PE as compared to normal pregnancies.