Normal Placental Function Research Articles

Spatial and temporal expression of folate-related transporters and metabolic enzymes during mouse placental development

It is well understood that maternal folate deficiency can cause abnormal fetal development. However, the extent to which placental development and function are also dependent upon folate uptake and metabolism remains unclear. To understand which trophoblast cell types may be affected by folate deficiency or abnormal folate metabolism, we completed a comprehensive spatial and temporal protein expression analysis of folate receptor (Folr), folate transporters (proton-coupled folate receptor [Slc46a1 or PCFT] and reduced folate carrier-1 [Rfc1]) and folate metabolic enzymes (5,10-methylenetetrahydrofolate reductase [Mthfr] and methionine synthase [Mtr]) in histological sections of mouse placentas from early development (E8.5) until term (E18.5). We observed that the highest level of protein expression was during early development (E8.5-E10.5), prior to the formation of the three main layers of the mature placenta suggesting that folate uptake and metabolism may be required for placental development, itself. As expected, the labyrinth trophoblast cells, which are responsible for nutrient transport, expressed these proteins throughout pregnancy, including robust expression in the sinusoidal trophoblast giant cells that line the maternal blood spaces. Other trophoblast giant cell (TGC) subtypes (parietal-TGCs and canal-TGCs), whose function does not include nutrient transport, expressed folate transporters and enzymes from E8.5 onwards. Remarkably, these proteins were also detected in glycogen trophoblast cells from E12.5–E18.5 suggesting a new role in folate uptake and metabolism for these cells. Together, these data provide evidence that folate may be necessary for normal placental development and function, and perturbations in its availability or metabolism may lead to secondary effects on fetal development.

Crim1 has an essential role in glycogen trophoblast cell and sinusoidal-trophoblast giant cell development in the placenta

Normal placental development and function is essential for fetal growth of eutherian mammals. Mutational studies have shown that numerous growth factors are required for placental development and differentiation of placental lineages. Here, using a gene-trap mutant mouse line, Crim1KST264, we show that Crim1 is essential for murine placental development. Crim1 is a developmentally expressed, trans-membrane regulator of growth factor activity. Crim1KST264/KST264 mutant placentae displayed hypoplasia from 13.5 dpc, and altered structure from 15.5 dpc, including alterations in cell number in both the junctional and labyrinth zones. Using the reporter gene from the Crim1KST264 allele, we found that Crim1 is expressed in multiple cell types of the placenta, including strong expression in the spongiotrophoblast cells of the junctional zone. In the junctional zone of Crim1KST264/KST264 placentae, there was an increase in the glycogen trophoblast cells adjacent to the spongiotrophoblast cells. In the labyrinth zone, we found a decrease in the density of sinusoidal-trophoblast giant cells. Our findings show that Crim1 is required for placental development, and is necessary for the proper differentiation of sinusoidal-trophoblast giant cells and glycogen trophoblast cells.

Understanding the Mechanism of Insulin and Insulin-Like Growth Factor (IGF) Receptor Activation by IGF-II

BackgroundInsulin-like growth factor-II (IGF-II) promotes cell proliferation and survival and plays an important role in normal fetal development and placental function. IGF-II binds both the insulin-like growth factor receptor (IGF-1R) and insulin receptor isoform A (IR-A) with high affinity. Interestingly both IGF-II and the IR-A are often upregulated in cancer and IGF-II acts via both receptors to promote cancer proliferation. There is relatively little known about the mechanism of ligand induced activation of the insulin (IR) and IGF-1R. The recently solved IR structure reveals a folded over dimer with two potential ligand binding pockets arising from residues on each receptor half. Site-directed mutagenesis has mapped receptor residues important for ligand binding to two separate sites within the ligand binding pocket and we have recently shown that the IGFs have two separate binding surfaces which interact with the receptor sites 1 and 2.Methodology/Principal FindingsIn this study we describe a series of partial IGF-1R and IR agonists generated by mutating Glu12 of IGF-II. By comparing receptor binding affinities, abilities to induce negative cooperativity and potencies in receptor activation, we provide evidence that residue Glu12 bridges the two receptor halves leading to receptor activation.Conclusions/SignificanceThis study provides novel insight into the mechanism of receptor binding and activation by IGF-II, which may be important for the future development of inhibitors of its action for the treatment of cancer.

OC18.05: Neurodevelopmental outcome at 24‐months of full‐term small‐for‐gestational age infants with normal placental function

OC18.05: Neurodevelopmental outcome at 24‐months of full‐term small‐for‐gestational age infants with normal placental function

W14.3 Increased incidence of pre-eclampsia in singleton pregnancies with breech presentation at delivery

Studies on the effects of various factors, including xenobiotics, on the maternal–fetal connections in the placenta are restricted by the lack of a simple and inexpensive research model. We used placentomes collected at a slaughterhouse to in vitro study the bovine sections contained integral maternal–fetal connections. The placentomes from cows (n = 4/experiment, 120–150 days post coitum) were cut using a razor blade into 60–80 mg sections and incubated in either DMEM/Ham's F-12 or M-199 supplemented with FCS (2%, 5% or 10%), amniotic fluid (AF or inactive AF, 10% or 20%) or both. The sections (n = 4/supplement) were incubated for 24 or 48 h in a water bath at 37.5 °C in an atmosphere of 5% CO2 and 95% O2. The structure and secretory activity of placentome sections were maintained when incubated in DMEM/Ham's F-12 with 2% FCS and 10% AF. M-199 was less acidified than DMEM/Ham's F-12 during incubation, and thus, this medium was better able to maintain the integrity of the placenta and the secretion of estradiol, progesterone and oxytocin for 48 h. Moreover, we detected a decrease in the expression of placenta-specific 1 (PLAC1) mRNA (an indicator of trophoblast proliferation) and an increase in the levels of keratin 8 (KRT8; a marker of normal placental barrier function) and hypoxia induced factor 1α (HIF1α; a marker of hypoxia) mRNA. These results indicate the presence of adaptation and repair mechanisms and confirm the biological activity of the placentome sections. We propose the use of placentome sections as an in vitro model to study maternal–fetal connections in cows.

Regulation of early trophoblast differentiation – Lessons from the mouse

The earliest stages of trophoblast differentiation are of tremendous importance to mediate implantation and to lay the anatomical foundations for normal placental development and function throughout gestation. Yet our molecular insights into these early developmental processes in humans have been limited by the inaccessibility of material and the unavailability of trophoblast cell lines that fully recapitulate the behaviour of early placental trophoblast. In this review we highlight recent advances that have come from the study of distinct stem cell types representative of the embryonic and extraembryonic lineages in the mouse, and from the study of mouse mutants. These models have revealed the presence of intricate transcriptional networks that are set up by signalling pathways, translating extracellular growth factor and cell positional information into distinct lineage-specific transcriptional programmes. The trophoblast specificity of these networks is ensured by epigenetic mechanisms including DNA methylation and histone modifications that complement each other to define trophoblast cell fate and differentiation. Despite the anatomical differences between mouse and human placentas, it seems that important aspects of early trophoblast specification are conserved between both species. Thus we may be able to build on our insights from the mouse to better understand early trophoblast differentiation in the human conceptus which is important for improving assisted reproductive technologies and may enable us in the future to derive human trophoblast stem cell lines. These advances will facilitate the investigation of genetic, epigenetic and environmental influences on early trophoblast differentiation in normal as well as in pathological conditions.

Editor’s Choice

Editor’s Choice

Down syndrome screening: imagining the screening test of the future

Prenatal screening for Down syndrome (DS) is performed by risk calculation based on biochemical and biometric parameters. This way, approximately 75–85% of all DS cases can be detected. A way to improve detection rates is to search for new screening markers. Since the majority of biomarkers used in current DS screening are predominantly produced by the placenta, and the presence of an extra chromosome (as in DS) complicates placental development and function, it is plausible to assume that new potential screening markers may also originate from the placenta. Any alterations in these markers can be attributed to abnormal placental development and function. This article focuses on normal early placental development and function compared with that in DS pregnancies. Using this knowledge, we reason towards candidate biomarkers that may be useful in screening for DS.

Beyond oxygen: complex regulation and activity of hypoxia inducible factors in pregnancy

In the first trimester the extravillous cytotrophoblast cells occlude the uterine spiral arterioles creating a low oxygen environment early in pregnancy, which is essential for pregnancy success. Paradoxically, shallow trophoblast invasion and defective vascular remodelling of the uterine spiral arteries in the first trimester may result in impaired placental perfusion and chronic placental ischemia and hypoxia later in gestation leading to adverse pregnancy outcomes. The hypoxia inducible factors (HIFs) are key mediators of the response to low oxygen. We aimed to elucidate mechanisms of regulation of HIFs and the role these may play in the control of placental differentiation, growth and function in both normal and pathological pregnancies. The Pubmed database was consulted for identification of the most relevant published articles. Search terms used were oxygen, placenta, trophoblast, pregnancy, HIF and hypoxia. The HIFs are able to function throughout all aspects of normal and abnormal placental differentiation, growth and function; during the first trimester (physiologically low oxygen), during mid-late gestation (where there is adequate supply of blood and oxygen to the placenta) and in pathological pregnancies complicated by placental hypoxia/ischemia. During normal pregnancy HIFs may respond to complex alterations in oxygen, hormones, cytokines and growth factors to regulate placental invasion, differentiation, transport and vascularization. In the ever-changing environment created during pregnancy, the HIFs appear to act as key mediators of placental development and function and thereby are likely to be important contributors to both normal and adverse pregnancy outcomes.

Neurobehavior in Term, Small-for-Gestational Age Infants With Normal Placental Function

The goal was to evaluate the neurobehavioral outcomes of term, small-for-gestational age (SGA) newborns with normal placental function. A cohort of consecutive term SGA newborns with normal prenatal umbilical artery Doppler ultrasound findings was created and compared with a group of term infants with size appropriate for gestational age, who were sampled from our general neonatal population. Neonatal behavior was evaluated at corrected age of 40 +/- 1 weeks with the Neonatal Behavioral Assessment Scale. The effect of the study group on each Neonatal Behavioral Assessment Scale area was adjusted, through multivariate analysis of covariance, for smoking during pregnancy, maternal BMI, socioeconomic level, onset of labor, mode of delivery, use of epidural anesthetic medication, gestational age at delivery, postnatal age (in days) at evaluation, and gender. A total of 202 newborns (102 SGA and 100 appropriate for gestational age) were included. All of the neurobehavioral areas studied were poorer in the SGA group, with significance for attention, habituation, motor, social-interactive, and regulation of state. The average mean differences in scores between the study groups were 0.77 (95% confidence interval: 0.38-1. 14) for attention, 0.64 (95% confidence interval: 0.13-1.14) for habituation, 0.52 (95% confidence interval: 0.31-0.74) for motor, 0.95 (95% confidence interval: 0.54-1.37) for social-interactive, and 0.68 (95% confidence interval: 0.23-1.13) for regulation of state. These differences remained significant after adjustment for potential confounders. Term SGA newborns with no signs of placental insufficiency had poorer neurobehavioral competencies, which suggests delayed neurologic maturation.

Expression of Adrenomedullin 2 (ADM2)/Intermedin (IMD) in Human Placenta: Role in Trophoblast Invasion and Migration1

Calcitonin gene-related peptide (CALCB), amylin, and adrenomedullin (ADM) belong to a unique group of calcitonin (CALCA)/CALCB family peptides that have overlapping biological effects owing to their structure and cross-reactivity between receptors. CALCB and ADM are expressed in fetoplacental tissues and are important in maintaining normal placental function. Recently, ADM 2 (ADM2)/intermedin was identified as a novel CALCA/CALCB family peptide that functions through CALCB and ADM receptors. ADM2 is expressed in the pituitary, digestive tract, and other organs of vertebrates and reduces blood pressure in both normal and hypertensive rats. We recently reported that the level of immunoreactive ADM2 is significantly upregulated in pregnant rats and that its hypotensive effects are also increased during rat pregnancy. Furthermore, infusion of ADM2 antagonist in pregnant rats causes fetoplacental growth restriction. The objective of this study was to analyze the expression and possible role of ADM2 in human placenta. We show that ADM2 mRNA is expressed in human placenta and that immunoreactive ADM2 is localized in syncytiotrophoblasts, cytotrophoblasts, and endothelial cells throughout human pregnancy. This study also demonstrates that ADM2 enhances the invasion and migration of first-trimester HTR-8SV/neo cells. ADM2 increases the invasive index of HTR-8SV/neo cells by 2.2-fold compared with controls. Taken together, the findings from this study suggest that ADM2 may have a role in the physiology of human pregnancy via regulation of trophoblast invasion and migration.

Cell cycle and apoptosis in normal and cloned bovine near-term placentae

The bovine maternal epithelium is composed of cuboidal cells interspersed with low columnar cells having centrally located nuclei. Bovine trophoblast is composed of two cell types: mononuclear trophoblastic and giant trophoblastic cells that can have two or more nuclei. Number of apoptotic cells and proliferative cells are variable in both cell populations. This study compared tissue growth and apoptosis by flow cytometry in the cell population found at distinct placental regions (central region of placentomes, ≤1-cm microplacentomes and the interplacentomal region) between normal and cloned near-term bovine pregnancies. After a morphological comparison between regions and groups (controls vs . clones), a lesser proportion of diploid to tetraploid cells was observed in the central region of placentomes and in microplacentomes from cloned-derived pregnancies. In addition, cloned animals had a fewer apoptotic cells in the central region of the placentome and in interplacentomal region and a greater proliferative capacity in all regions (cells in G 2/M) near term as opposed to control animals. These results may reveal the existence of a relationship between such changes in the proportions of uterine and trophoblastic epithelial cells at the end of pregnancy and normal placental function. This could be related to faulty placentation in early pregnancy, placental insufficiency during pregnancy or lack of placental and/or fetal maturation in late pregnancy, which may contribute to some of the abnormalities after in vitro embryo manipulations, such as poor preparation and initiation of parturition, prolonged gestation and lesser post-natal survival in some cloned animals.

Defect of Hepatocyte Growth Factor Activator Inhibitor Type 1/Serine Protease Inhibitor, Kunitz Type 1 (Hai-1/Spint1) Leads to Ichthyosis-Like Condition and Abnormal Hair Development in Mice

Hepatocyte growth factor activator inhibitor type 1 (HAI-1)/serine protease inhibitor, Kunitz type 1 (SPINT1) is a membrane-bound, serine proteinase inhibitor initially identified as an inhibitor of hepatocyte growth factor activator. It also inhibits matriptase and prostasin, both of which are membrane-bound serine proteinases that have critical roles in epidermal differentiation and function. In this study, skin and hair phenotypes of mice lacking the Hai-1/Spint1 gene were characterized. Previously, we reported that the homozygous deletion of Hai-1/Spint1 in mice resulted in embryonic lethality attributable to impaired placental development. To test the role of Hai-1/Spint1 in mice, the placental function of Hai-1/Spint1-mutant mice was rescued. Injection of Hai-1/Spint1(+/+) blastocysts with Hai-1/Spint1(-/-) embryonic stem cells successfully generated high-chimeric Hai-1/Spint1(-/-) embryos (B6Hai-1(-/-High)) with normal placentas. These embryos were delivered without apparent developmental abnormalities, confirming that embryonic lethality of Hai-1/Spint1(-/-) mice was caused by placental dysfunction. However, newborn B6Hai-1(-/-High) mice showed growth retardation and died by 16 days. These mice developed scaly skin because of hyperkeratinization, reminiscent of ichthyosis, and abnormal hair shafts that showed loss of regular cuticular septation. The interfollicular epidermis showed acanthosis with enhanced Akt phosphorylation. Immunoblot analysis revealed altered proteolytic processing of profilaggrin in Hai-1/Spint1-deleted skin with impaired generation of filaggrin monomers. These findings indicate that Hai-1/Spint1 has critical roles in the regulated keratinization of the epidermis and hair development.

Specific tumour-associated methylation in normal human term placenta and first-trimester cytotrophoblasts

Human placentation displays many similarities with tumourigenesis, including rapid cell division, migration and invasion, overlapping gene expression profiles and escape from immune detection. Recent data have identified promoter methylation in the Ras association factor and adenomatous polyposis coli tumour suppressor genes as part of this process. However, the extent of tumour-associated methylation in the placenta remains unclear. Using whole genome methylation data as a starting point, we have examined this phenomenon in placental tissue. We found no evidence for methylation of the majority of common tumour suppressor genes in term placentas, but identified methylation in several genes previously described in some human tumours. Notably, promoter methylation of four independent negative regulators of Wnt signalling has now been identified in human placental tissue and purified trophoblasts. Methylation is present in baboon, but not in mouse placentas. This supports a role for elevated Wnt signalling in primate trophoblast invasiveness and placentation. Examination of invasive choriocarcinoma cell lines revealed altered methylation patterns consistent with a role of methylation change in gestational trophoblastic disease. This distinct pattern of tumour-associated methylation implicates a coordinated series of epigenetic silencing events, similar to those associated with some tumours, in the distinct features of normal human placental invasion and function.

Divergent Effects of Ephedrine and Phenylephrine on Cardiovascular Hemodynamics of Near-Term Fetal Sheep Exposed to Hypoxemia and Maternal Hypotension

Erkinaro, T.; Mäkikallio, K.; Acharya, G.; Päkkilä, M.; Kavasmaa, T.; Huhta, J.C.; Alahuhta, S.; Räsänen, J. Author Information

Ultrasound of the Placenta

Abstract This review covers ultrasound evaluation of the normal and abnormal placenta with clinical correlation. Normal placental function is essential for a healthy pregnancy outcome as well as for maternal, fetal, childhood, and adult health. Abnormal placental function may result in a compromised pregnancy, creating pathology for the fetus and mother alike. Despite the fact that placental anatomy, function, and location has far-reaching effects for the parents and the developing offspring, ultrasound examination of the placenta is often considered secondary to the fetus by expectant parents and sonographers as well. Location, size, shape, and architecture are easily ascertained with two-dimensional techniques. Three-dimensional ultrasound and Doppler techniques have opened up the frontier of placental function and have set the stage to make placental evaluation as fascinating as that of the fetus. Learning objectives To assess normal placenta by ultrasound. To discuss abnormal placenta and umbilical cord. To understand placentation in multiple gestation.

Umbilical artery half peak systolic velocity deceleration time throughout pregnancy and its role in fetuses with bradycardia

To describe a new technique for Doppler waveform quantification of the fetal umbilical artery, the half peak systolic velocity (h-PSV) deceleration time, which measures the time that it takes a single flow velocity waveform to halve its maximum systolic velocity. As this measurement is independent of fetal heart rate, its role in the evaluation of placental vascular resistance in fetuses with bradycardia was also explored. The umbilical artery impedance indices and the h-PSV deceleration time were measured in 532 normal singleton fetuses from 17 to 41 weeks of gestation. A nomogram was established and its usefulness in assessing fetuses with bradycardia was evaluated. The relationship between umbilical artery h-PSV deceleration time and gestational age was best described by a linear formula (y = 6.5523x + 12.41; R(2) = 0.6346), h-PSV deceleration time increasing by 6.6 ms per week. The correlation coefficients for the 95(th), 50(th) and 5(th) centiles were 0.97, 0.98 and 0.98, respectively. Measurement of h-PSV deceleration time was reproducible, with interobserver and intraobserver variabilities of 10.7% and 7.4%, respectively. Among fetuses with bradycardia (n = 7), the h-PSV deceleration time was above the 5(th) centile in all cases, suggesting normal placental function in spite of abnormally increased impedance indices obtained with traditional Doppler indices in six. The h-PSV deceleration time increases linearly during the second and third trimesters of pregnancy. As its measurement is reproducible and independent of heart rate, it may be useful in the assessment of well-being in fetuses with bradycardia.

Divergent effects of ephedrine and phenylephrine on cardiovascular hemodynamics of near‐term fetal sheep exposed to hypoxemia and maternal hypotension

We hypothesized that the administration of ephedrine and phenylephrine for maternal hypotension modifies cardiovascular hemodynamics in near-term sheep fetuses. At 115-136 days of gestation, chronically instrumented, anesthetized ewes with either normal placental function or increased placental vascular resistance after placental embolization were randomized to receive boluses of ephedrine (n = 12) or phenylephrine (n = 12) for epidural-induced hypotension after a short period of hypoxemia. Fetal cardiovascular hemodynamics were assessed by Doppler ultrasonography at baseline, during hypotension and after vasopressor treatment. During hypotension, fetal PO(2) decreased and proximal branch pulmonary arterial and pulmonary venous vascular impedances increased. Additionally, in the embolized fetuses, the time-velocity integral ratio between the antegrade and retrograde blood flow components of the aortic isthmus decreased. These parameters were restored to baseline conditions by ephedrine but not by phenylephrine. With phenylephrine, weight-indexed left ventricular cardiac output and ejection force decreased in the non-embolized fetuses, and the proportion of isovolumetric contraction time of the total cardiac cycle was elevated in the embolized fetuses. After exposure to hypoxemia and maternal hypotension, ephedrine restored all fetal cardiovascular hemodynamic parameters to baseline. Phenylephrine did not reverse fetal pulmonary vasoconstriction or the relative decrease in the net forward flow through the aortic isthmus observed in fetuses with increased placental vascular resistance. Moreover, fetal left ventricular function was impaired during phenylephrine administration.

Maternal levels of free fetal DNA are elevated in pregnancies with growth restriction due to placental dysfunction

Objective. Fetal growth restriction (FGR) is associated with an increased risk of perinatal mortality and morbidity but can have many different causes. Non-cellular fetal DNA in maternal blood offers many opportunities for noninvasive prenatal diagnosis. It is likely that the source of the DNA is apoptosis or cell death in the placenta and that free fetal DNA (ffDNA) levels could theoretically be increased in placental dysfunction or infarction. We hypothesised that non-cellular fetal DNA levels would be increased only in the subset of FGR cases with placental dysfunction, which could explain previous contradictory reports. Methods. We used plasma samples obtained during a previous study from pregnant women with singleton male pregnancies as controls and from women with small-for-gestational-age (SGA) infants that had been classified as having FGR due to placental dysfunction. A third group was defined as normal but small fetuses with placental function within the normal ranges indicated by Doppler studies. Twenty-two cases were identified in the third trimester of pregnancy (8 from the control group and 7 from each of the FGR and the normal small groups). DNA was extracted and the DYS14 gene of the Y chromosome was quantified by real-time quantitative polymerase chain reaction (PCR). Results. ffDNA levels were higher in pregnancies with FGR due to placental dysfunction than in either normal pregnancies or those with SGA fetuses from causes other than placental dysfunction. There was no significant difference in the ffDNA levels between the fetuses of normal growth and those with FGR from other causes. Conclusion. The level of ffDNA in maternal plasma is increased in pregnancies complicated by FGR secondary to placental dysfunction but not in those with small fetuses with normal placental function. South African Journal of Obstetrics and Gynaecology Vol. 13 (2) 2007: pp. 60-63

2005 Trophoblast Research Award Lecture: Defects in the Keratin Cytoskeleton Disrupt Normal Murine Placental Development and Trophoblast Cell Function

The keratin cytoskeleton is present in all trophoblast cell subtypes of the mouse and human placenta and is required to maintain the structural integrity of these cells. Recently, various genetic mouse models have shown that a normal keratin network is necessary for placental development. Keratin-deficiency leads to trophoblast giant cell fragility, breaking the barrier between the conceptus and the maternal blood circulation. Alternatively, keratin aggregation prevents chorioallantoic attachment, a key developmental milestone required for the formation of the labyrinth within the mouse placenta. These models give us insight into cytokeratin function in human trophoblast cell subtypes and suggest that defects in the keratin cytoskeleton may result in intrauterine growth restriction or miscarriage.