IFPA Trophoblast Research Award Lecture: the dynamic role of Bcl-2 family members in trophoblast cell fate.

Bcl-2 family members are important regulators of cell fate in normal organ development and in disease status. Pro- and anti-apoptotic members of this family function through a complex network of homo- and hetero-dimers to determine whether a cell lives or dies. Members of the Bcl-2 family are classically recognized for their role in apoptosis, yet emerging evidence has highlighted their importance in the regulation of cell cycle. Cellular proliferation, differentiation and death accompany early placental development of the trophoblast lineage. We have recently reported on the expression and function of two Bcl-2 family members in normal placental development, namely the pro-apoptotic Mtd/Bok, and its anti-apoptotic partner Mcl-1 and have found that their expression is upregulated by low oxygen, a key mediator of trophoblast cell proliferation in early placentation. Interestingly, we have also reported that the expression of the Mtd/Mcl-1 system is altered in preeclampsia, a placental pathology associated with a status of oxidative stress and typically characterized by an immature proliferative trophoblast phenotype and excessive trophoblast cell death. In this pathology levels of pro-apototic Mtd-L and Mtd-P are increased and anti-apoptotic Mcl-1 is cleaved in to a pro-apoptotic isoform. Disruption in Mtd/Mcl-1 expression seen in preeclampsia may contribute to both the increased apoptosis and hyperproliferative nature of this disorder.

Inhibition of TGF-beta 3 restores the invasive capability of extravillous trophoblasts in preeclamptic pregnancies.

Preeclampsia, the major cause of maternal morbidity and mortality in developed countries, is associated with abnormalities of placenta function due to shallow invasion of the maternal decidua by trophoblasts. Data suggest that TGF-beta may play a role in inhibiting trophoblast outgrowth or invasion, or both. We report that placental TGF-beta 3 expression is high in early pregnancy but falls at around 9 weeks' gestation. This pattern is inversely correlated with trophoblast outgrowth and fibronectin synthesis, markers of early trophoblast differentiation toward an invasive phenotype. We demonstrate that TGF-beta 3 is overexpressed in preeclamptic placentae. In contrast to control placentae, explants from preeclamptic pregnancies fail to exhibit spontaneous invasion in vitro. Significantly, antisense-induced inhibition of TGF-beta 3 expression, and inhibition of TGF-beta 3 activity with antibodies, induces the formation of columns of trophoblast cells, which migrate out of the explant into the underlying Matrigel. To our knowledge, this is the first demonstration that the hypoinvasive placental phenotype characteristic of preeclampsia can be essentially normalized in vitro by biochemical manipulation. We speculate that a failure to downregulate expression of TGF-beta 3 at around 9 weeks' gestation results in shallow trophoblast invasion and predisposes the pregnancy to preeclampsia.

Hypoxia-inducible factor-1 mediates the biological effects of oxygen on human trophoblast differentiation through TGFbeta(3)

During early pregnancy, placentation occurs in a relatively hypoxic environment that is essential for appropriate embryonic development. Intervillous blood flow increases around 10 to 12 weeks of gestation and results in exposure of trophoblast cells to increased oxygen tension. Before this time, low oxygen appears to prevent trophoblast differentiation toward an invasive phenotype. Using human villous explants of 5-8 weeks' gestation, we found that low oxygen tension triggered trophoblast proliferation, fibronectin synthesis, alpha(5) integrin expression, and gelatinase A activity. These biochemical markers were barely detectable under oxic conditions. We therefore examined the placental expression of hypoxia-inducible factor-1 (HIF-1), a master regulator of oxygen homeostasis, and determined that expression of HIF-1alpha subunit during the first trimester of gestation parallels that of TGFbeta(3), an inhibitor of extravillous trophoblast differentiation. Expression of both molecules is high in early pregnancy and falls around 9 weeks of gestation, when placental pO(2) levels are believed to increase. Increasing oxygen tension induced a similar decrease in expression in cultured explants. Moreover, antisense inhibition of HIF-1alpha expression in hypoxic explants inhibited expression of TGFbeta(3), arrested cell proliferation, decreased alpha(5) expression and gelatinase A activity, and triggered biochemical markers of an invasive trophoblast phenotype such as alpha(1) integrin and gelatinase B expression. These data suggest that the oxygen-regulated early events of trophoblast differentiation are in part mediated by TGFbeta(3) through HIF-1 transcription factors.

A novel Mtd splice isoform is responsible for trophoblast cell death in pre-eclampsia.

Pre-eclampsia is a serious disorder of human pregnancy, characterized by decreased utero-placental perfusion and increased trophoblast cell death. Presently, the mechanisms regulating trophoblast cell death in pre-eclampsia are not fully elucidated. Herein, we have identified a novel Mtd/Bok splice isoform (Mtd-P) resulting from exon-II skipping. Mtd-P expression was unique to early-onset severe pre-eclamptic placentae as assessed by quantitative real-time-PCR and immunoblotting. Mtd-P overexpression in cell lines (BeWo: cytotrophoblast-derived; and CHO: ovary-derived) resulted in increased apoptotic cell death as assessed by caspase-3 cleavage, internucleosomal DNA laddering and mitochondrial depolarization. Moreover, Mtd-P expression increased under conditions of low oxygenation/oxidative stress in human villous explants. Antisense knockdown of Mtd under conditions of oxidative stress resulted in decreased caspase-3 cleavage. We conclude that under conditions of reduced oxygenation/oxidative stress, Mtd-P causes trophoblast cell death in pre-eclampsia and hence may contribute to the molecular events leading to the clinical manifestations of this disease.

IFPA meeting 2015 workshop report III: nanomedicine applications and exosome biology, xenobiotics and endocrine disruptors and pregnancy, and lipid.

Workshops are an important part of the IFPA annual meeting, as they allow for discussion of specialized topics. At the IFPA meeting 2015 there were twelve themed workshops, three of which are summarized in this report. These workshops were related to various aspects of placental biology but collectively covered areas of pregnancy pathologies and placental metabolism: 1) nanomedicine applications and exosome biology; 2) xenobiotics and endocrine disruptors and pregnancy; 3) lipid mediators and placental function.

Human placental explants in culture: approaches and assessments.

Placental explant cultures in vitro are useful for studying tissue functions including cellular uptake, production and release of secretory components, cell interactions, proliferation, growth and differentiation, gene delivery, pharmacology, toxicology, and disease processes. A variety of culture conditions are required to mimic in utero environments at different times of gestation including differing oxygen partial pressures, extracellular matrices and culture medium. Optimization of explant methods is examined for first and third trimester human placental tissue and the biological processes under investigation.

Activin is a local regulator of human cytotrophoblast cell differentiation.

Cytotrophoblast cells of the first trimester placenta are highly invasive when removed from villi and cultured in vitro. In vivo, however, only selected cytotrophoblast cells break through the overlying syncytiotrophoblast to form cytotrophoblast columns and ultimately invade the endometrium. To explore the role of paracrine growth factors in regulating cytotrophoblast development, we cultured explants of first trimester chorionic villi in vitro. Both activin and inhibin, as well as the activin binding protein follistatin, are produced by various trophoblast cells throughout pregnancy. We found that addition of activin-A, but not inhibin-A, stimulated the outgrowth of cytotrophoblast cells into the surrounding matrix. This outgrowth was characteristic of that observed in extravillous cytotrophoblast cells in vivo; it was accompanied by cell division within the proximal region of the cytotrophoblast outgrowth, synthesis of fibronectin, as well as the expression of markers characteristic of invasive cytotrophoblast cells, human leukocyte antigen-G and matrix metalloproteinase (MMP)-9. Activin also specifically induced the early expression of MMP-2 within villous cytotrophoblast cells. Addition of the activin binding protein, follistatin, blocked all of the effects of exogenous activin. The morphological and biochemical effects of activin were similar to those observed when signaling of endogenous transforming growth factor-beta was blocked. Interestingly, the latter effects were also reversed by the addition of follistatin. These data suggest that activin plays a local role in promoting cytotrophoblast column formation, likely by regulating the differentiation of villous cytotrophoblast into extravillous cytotrophoblast cells.

Endoglin regulates trophoblast differentiation along the invasive pathway in human placental villous explants.

Successful invasion of the maternal vascular system by trophoblast cells is a prerequisite for the establishment of a normal hemochorial placenta. Transforming growth factor-beta (TGFbeta) has been implicated in the regulation of trophoblast invasiveness into the uterus. Endoglin is a component of the TGFbeta receptor complex that binds beta1 and beta3 isoforms and is expressed at high levels on syncytiotrophoblast throughout pregnancy and is also transiently up-regulated on extravillous trophoblasts differentiating along the invasive pathway. We investigated the role of endoglin in a serum-free human villous explant culture system that allows the study of trophoblast outgrowth, migration, and invasion and mimics events occurring in anchoring villi during the first trimester of gestation. Addition to explant cultures from 5-8 weeks gestation of a monoclonal antibody to endoglin or of antisense endoglin oligonucleotides significantly stimulated trophoblast outgrowth and migration. These responses were specific, as incubation of explants with nonimmune IgG or sense and scrambled oligonucleotides had no effect. Antisense endoglin-induced trophoblast outgrowth and migration were accompanied by cell division of villous-associated trophoblasts within the proximal region of the forming column and by the characteristic switch in integrins observed in anchoring villi in situ. Treatment of villous explants with antibody and antisense oligonucleotides to endoglin also resulted in an increased fibronectin release into the culture medium. The stimulatory effect of antisense endoglin on fibronectin production was overcome by the addition of exogenous TGFbeta2, but not TGFbeta1 and -beta3. These findings suggest that endoglin expression in the transition from polarized to nonpolarized trophoblasts in anchoring villi is necessary for mediation of the inhibitory effect of TGFbeta1 and/or TGFbeta3 on trophoblast differentiation along the invasive pathway.

Oxygen regulation of placental 11 beta-hydroxysteroid dehydrogenase 2: physiological and pathological implications.

Preeclampsia (PE) is a major cause of maternal and perinatal morbidity and mortality. The genesis of PE is related to deficient trophoblast invasion of maternal spiral arteries, which might result in a reduction of placental (PL) oxygen (O(2)). An absence of increased O(2) that normally occurs around the 10-12th wk of gestation results in aberrant expression of genes that might contribute to the pathophysiology of PE. We examined the expression and regulation of PL 11 beta-hydroxysteroid dehydrogenase 2 (11 beta-HSD) in normal pregnancies and in PE. Two types of 11 beta-HSD exist in the placenta, 11 beta-HSD1 and 11 beta-HSD2. 11 beta-HSD2 is thought to protect the fetus from cortisol excess. In PE, both the expression and activity of PL 11 beta-HSD2 were reduced significantly compared with those in age-matched controls. As PE is associated with a reduction of PL O(2), we next investigated whether in normal pregnancy 11 beta-HSD2 expression changes at the time of the increase in O(2). 11 beta-HSD2 was detected as early as 5 wk, with expression limited to the syncytiotrophoblast (ST). At 10-12 wk, this expression increased and was also found in the cytotrophoblast and extravillous trophoblast. These results were substantiated by Western blot. The ability of O(2) to regulate 11 beta-HSD2 was determined both in cultures of villous explant from early gestation and in term trophoblast cells after incubation under 3% or 20% O(2). Villous explants cultured under 20% O(2) showed higher enzyme activity and expression compared with 3% O(2). Term trophoblast cells also exhibited higher enzyme activity at 20% vs. 3% O(2). No change in 11 beta-HSD1 expression was observed in early pregnancy or in PE. This is the first report to suggest that 11 beta-HSD2 is O(2) dependent in first and third trimester placenta during human gestation.

Adriana and Luisa Castellucci Award lecture 2001. Hypoxia inducible factor-1: oxygen regulation of trophoblast differentiation in normal and pre-eclamptic pregnancies--a review.

During early pregnancy, trophoblast differentiation occurs in an environment of relative low oxygen tension which is essential for normal embryonic and placental development. At around 10-12 weeks' gestation, when the intervillous space opens to maternal blood, there is an increase in Po(2). This increase correlates with the time of maximal trophoblast invasion into the maternal decidua, which allows extravillous trophoblast cells to access and remodel the maternal spiral arteries. Hypoxia Inducible Factor 1(HIF-1) is a transcription factor which activates gene transcription in response to varying oxygen concentration of cells. HIF-1 is a heterodimer composed of the inducible HIF-1alpha and the constitutively expressed HIF-1beta/ARNT subunits. Using villous explants, we have demonstrated that the oxygen-regulated events of early trophoblast differentiation are in part mediated by TGFbeta(3), an inhibitor of trophoblast differentiation, via HIF-1alpha. Pre-eclampsia is a disease of pregnancy that is characterized by shallow trophoblast invasion. Recently, we have reported that TGFbeta(3) is over-expressed in pre-eclamptic pregnancy and that its down-regulation restores invasive capability to trophoblast cells. Because TGFbeta(3) is downstream of HIF-1alpha, in the present study we investigated the expression of HIF-1alpha in pre-eclamptic placentae and age-matched controls using in situ hybridization and histochemical analyses. We found that HIF-1alpha mRNA and protein expression are abnormally elevated in pre-eclamptic placental tissue when compared to normal placental tissue. We conclude that pre-eclampsia may result from a developmental failure of oxygen to increase or of trophoblast cells to respond and/or sense an increase in oxygen. This will prevent the normal TGFbeta3 down-regulation and will lead to poor trophoblast invasion predisposing the pregnancy to pre-eclampsia.

Mechanisms of oxygen sensing in human trophoblast cells.

During pregnancy, changes in oxygen tension are essential for proper embryonic and placental development. Little is known about the mechanisms underlying mammalian cellular adaptations to changes in oxygen tension. Currently, we have explored putative mechanisms by which human trophoblast cells may sense oxygen. In order to investigate a role for hemoproteins in oxygen sensing, we cultured human villous explants of 5-8 weeks gestation under 20 per cent O(2) in the presence of either cobalt chloride or desferrioxamine, which interfere with the ability of iron (heme) to interact with oxygen. Treatment with these compounds mimicked hypoxia by stimulating the low oxygen effect on extravillous trophoblast outgrowth (EVT) and inducing HIF-1alpha expression, analogous to that observed in explants cultured at 3 per cent O(2). Addition of unhindered iron, in the form of iron chloride, to the treated-explants reversed the stimulatory effect on EVT outgrowth and HIF-1alpha expression. Subsequently, in order to probe into a mitochondrial role in trophoblast oxygen sensing, we cultured first trimester villous explants under 3 per cent O(2) in the presence of either diphenyleneiodonium or rotenone, known inhibitors of flavin-containing proteins. Treated-explants showed inhibition of the typical low oxygen-induced EVT outgrowth, when compared to untreated controls. Thus, this data supports a hypothesis that trophoblast cells may utilize mitochondria and/or hemoproteins as oxygen sensors to detect the critical changes in oxygen tension during pregnancy.

Oxygen and placental development during the first trimester: implications for the pathophysiology of pre-eclampsia.

During early pregnancy, placentation occurs in a relatively hypoxic environment which is essential for appropriate embryonic development. Intervillous blood flow increases at around 10-12 weeks of gestation and results in exposure of the trophoblast to increased oxygen tension (PO2). Prior to this time, low oxygen appears to prevent trophoblast differentiation towards an invasive phenotype. In other mammalian systems, oxygen tension effects are mediated by hypoxia inducible factor-1 (HIF-1). We found that the ontogeny of HIF-1alpha subunit expression during the first trimester of gestation parallels that of transforming growth factor-beta3 (TGFbeta3), an inhibitor of early trophoblast differentiation. Expression of both molecules is high in early pregnancy and falls at around 10 weeks of gestation when placental PO2 levels are believed to increase. Antisense-induced inhibition of HIF-1alpha inhibited the expression of TGFbeta3, and stimulated extravillous trophoblast (EVT) outgrowth and invasion. Of clinical significance we found that TGFbeta3 expression was increased in pre-eclamptic placentae when compared to age-matched controls. Significantly, inhibition of TGFbeta3 by antisense oligonucleotides or antibodies restored the invasive capability to the trophoblast cells in pre-eclamptic explants. We speculate that if oxygen tension fails to increase, or trophoblasts do not detect this increase, HIF-1alpha and TGFbeta3 expression remain high, resulting in shallow trophoblast invasion and predisposing the pregnancy to pre-eclampsia. Effective fetal-maternal interactions during early placentation are critical for a successful pregnancy. Optimal placental perfusion requires the controlled invasion of trophoblast cells deep into the decidua to the spiral arteries. Trophoblast stem cells, also referred to as cytotrophoblast cells, reside in chorionic villi of two types, floating and anchoring villi. Floating villi, which represent the vast majority of chorionic villi, are bathed in maternal blood and primarily perform gas and nutrient exchange for the developing embryo. During early placentation, cytotrophoblast cells in the floating villi proliferate and differentiate by fusing to form the multinucleate syncytiotrophoblast layer. Cytotrophoblast cells in anchoring villi either fuse to form the syncytiotrophoblast layer, or break through the syncytium at selected sites and form multilayered columns of non-polarized extravillous trophoblast cells, which physically connect the embryo to the uterine wall (Figure 1). The extravillous trophoblast cells invade into the uterine wall as far as the first third of the myometrium and its associated spiral arteries, where they disrupt the endothelium and the smooth muscle layer and replace the vascular wall. This results in the conversion of the narrow calibre arteries into distended uteroplacental arteries, thereby increasing blood flow to the placenta and allowing an adequate supply of oxygen and nutrients to the growing fetus. The invasive activity of the extravillous trophoblast cells is at a maximum during the first trimester of gestation, peaking at around 10-12 weeks and declining thereafter. Insufficient invasion contributes to the development of pre-eclampsia, which often results in fetal intrauterine growth restriction, maternal hypertension and proteinuria. In contrast, unrestricted invasion is associated with premalignant conditions, such as invasive mole, and with malignant choriocarcinoma. Invading trophoblast cells undergo striking and rapid changes in cellular functions that are temporally and spatially regulated along the invasive pathway (Figure 1) (Cross, Werb and Fisher, 1994. The formation of the anchoring villi is accompanied by changes in synthesis and degradation of extracellular matrix proteins and their receptors, and changes in the spatial distribution of extracellular matrix proteins, as well as changes in the expression of adhesion molecules (Damsky, Fitzgerald and

TGF-beta 3 expression during umbilical cord development and its alteration in pre-eclampsia.

Members of the TGF-beta family have been shown to play an important role in numerous tissues during development. In the present study we have investigated the spatial and temporal expression of TGF-beta 3, in human umbilical cord development. Total TGF-beta 3 protein content, assessed by immunoblotting, increased with advancing gestation as did immunostaining and mRNA in Wharton's jelly fibroblasts. Immunohistochemical analysis revealed that TGF-beta 3 was present in all cell types. Temporal changes in TGF-beta 3 expression were observed in the vascular smooth muscle cells, such that with advancing gestation TGF-beta 3 protein expression and became mostly restricted to the extracellular compartment of the vascular media. This was associated with a decrease in TGF-beta 3 mRNA expression in umbilical vascular smooth muscle cells. Of clinical significance, umbilical cords from pregnancies complicated by pre-eclampsia, showed a significant reduction in total TGF-beta 3 protein expression when compared to those of age-matched patients. Both TGF-beta 3 mRNA and protein expression were downregulated in the endothelium and smooth muscle layers of the umbilical arteries, as well as in the Wharton jelly fibroblasts. Our data demonstrate that during umbilical cord development TGF-beta 3 expression is spatially and temporally regulated and that TGF-beta 3 expression is altered in umbilical cords of pregnancies complicated by pre-eclampsia. We speculate that the downregulation of TGF-beta 3 expression found in pre-eclamptic umbilical cord may contribute to the abnormal structure and mechanical properties seen in these pathological umbilical cords.

Hypoxia favours necrotic versus apoptotic shedding of placental syncytiotrophoblast into the maternal circulation.

In the third trimester of normal pregnancy, the mother tolerates daily shedding of several grams of dying placental trophoblast into the maternal circulation. The balance between apoptotic and necrotic shedding is presently unknown. Since pre-eclampsia is characterized by an altered placental oxygenation and increased trophoblast shedding, we investigated the role of oxygen on the balance of apoptotic versus necrotic trophoblast shedding in vitro. We studied human trophoblast turnover in explanted villi from late first and third trimester placentas in low oxygen (2 per cent) and higher oxygen tensions (6 per cent and 18 per cent) for up to 72h. Trophoblast turnover including apoptosis and necrosis were assessed by histology, immunolocalization of Mib-1 (proliferation marker), Bcl-2 (apoptosis inhibitor), activated caspase 3 (apoptosis promoter), cytokeratin 18 neo-epitope formation (M30 antibody), TUNEL test (DNA degradation), and (3)H-cytidine and(3) H-uridine incorporations. Culture in 2 per cent oxygen increased cytotrophoblast proliferation and syncytiotrophoblast shedding by necrosis. The proteins necessary for execution of apoptosis were mostly retained in the cytotrophoblast due to lack of syncytial fusion. Culture in 6 per cent and 18 per cent oxygen reduced cytotrophoblast proliferation. Syncytial fusion occurred and activity of caspase 3 was found in the syncytiotrophoblast; the latter remained intact demonstrating physiologic turnover, including apoptotic shedding. We conclude that severe placental hypoxia favours necrotic rather than apoptotic shedding of syncytial fragments into the maternal circulation. Since uteroplacental ischaemia is a significant risk factor for pre-eclampsia, these findings may explain the link between reduced uteroplacental blood flow and the systemic clinical manifestations of this disease.

Developmental differences in the expression of FGF receptors between human and mouse embryos.

INTRODUCTION: Fibroblast growth factor (FGF) signaling is essential for early trophoblast expansion and maintenance in the mouse, but is not required for trophectoderm specification during blastocyst formation. This signaling pathway is stably activated to expand the trophoblast stem cell compartment in vivo, while in vitro, FGFs are used for the derivation of trophoblast stem (TS) cells from blastocysts and early post-implantation mouse embryos. However, the function of FGFs during human trophoblast development is not known. METHODS: We sought to derive TS cells from human blastocysts in a number of culture conditions, including in the presence of FGFs and stem cell factor (SCF). We also investigated the expression of FGF receptors (FGFRs) in blastocysts, and the expression of FGFR2 and activated ERK1/2 in first trimester human placentae. RESULTS: We found that SCF, but not FGF2/4, improved the quality of blastocyst outgrowths, but we were unable to establish stable human TS cell lines. We observed CDX2 expression in the trophectoderm of fully blastocysts, but rarely observed transcription of FGFRs. FGFR2 protein was not detected in human blastocysts, but was strongly expressed in mouse blastocysts. However, we found robust FGFR2 expression and activated ERK1/2 in the cytotrophoblast layer of early human placenta. DISCUSSION: Our data suggests that initiation of FGF-dependent trophoblast expansion may occur later in human development, and is unlikely to drive maintenance of a TS cell compartment during the peri-implantation period. These findings suggest that cytotrophoblast preparations from early placentae may be a potential source of FGF-dependent human TS cells.

The double life of MULE in preeclamptic and IUGR placentae.

The E3 ubiquitin ligase MULE (Mcl-1 Ubiquitin Ligases E3) targets myeloid cell leukemia factor 1 (Mcl-1) and tumor suppressor p53 for proteasomal degradation. Although Mcl-1 and p53 have been implicated in trophoblast cell death in preeclampsia (PE) and intrauterine growth restriction (IUGR), the mechanisms regulating their expression in the human placenta remains elusive. Herein, we investigated MULE's involvement in regulating Mcl-1 and p53 degradation during normal and abnormal (PE, IUGR) placental development. MULE expression peaked at 5-7 weeks of gestation, when oxygen tension is low and inversely correlated with that of Mcl-1 and p53. MULE efficiently bound to Mcl-1 and p53 and regulated their ubiquitination during placental development. Exposure of first trimester villous explants to 3% O(2) resulted in elevated MULE expression compared with 20% O(2). Low-oxygen-induced MULE expression in JEG3 choriocarcinoma cells was abolished by hypoxia-inducible factor (HIF)-1α siRNA. MULE was overexpressed in both PE and IUGR placentae. In PE, MULE preferentially targeted p53 for degradation, allowing accumulation of pro-apoptotic Mcl-1 isoforms. In IUGR, however, MULE targeted pro-survival Mcl-1, allowing p53 to accumulate and exert its apoptotic function. These data demonstrate that oxygen regulates Mcl-1 and p53 stability during placentation via HIF-1-controlled MULE expression. The different preferential targets of MULE in PE and IUGR placentae classify early-onset PE and IUGR as distinct molecular pathologies.

Sex-specific differences in placental global gene expression in pregnancies complicated by asthma.

BACKGROUND: Chronic maternal asthma is associated with reduced growth of the female fetus and normal growth of the male fetus. The mechanisms that control the differential effects of maternal asthma on the fetus have not been fully elucidated but alterations in placental function may play a role. In the current study we have used microarray platform to examine fetal sex-specific global changes in placental gene expression in pregnancies complicated by asthma as compared to non-asthmatic subjects. METHODS: Placental RNA was extracted from 11 control subjects and 38 asthmatic subjects. Labeled cDNA was hybridized to an oligonucleotide chip with 1700 double spotted well-characterized human genes. Global gene expression data analysis and visualization were performed using the Binary Tree-Structured Vector Quantization (BTSVQ) software. Functional relationships of differentially expressed genes were assessed using protein-protein interaction database I2D, network analysis and visualization software NAViGaTOR and Ingenuity Pathway Analysis software. RESULTS: Overall, 65 genes were found to be altered in placentae of pregnancies complicated by asthma. Of these, only 6 genes were altered in male placentae. There were 59 gene changes in female placentae of asthmatic mothers relative to control placentae. Some of the sex-specific genes were associated with growth, inflammation and immune pathways. CONCLUSION: There are sex-specific alterations in placental gene expression in the presence of maternal asthma. Given that many of the identified genes in the female placentae were associated with or involved in cellular growth and tissue development, these may contribute to the sexually dimorphic difference in fetal growth in response to maternal asthma.

Review: Feto-placental vascularization: a multifaceted approach.

Doppler Ultrasound allows the in vivo study of feto-placental hemodynamics. Doppler flow velocity waveforms (FVW's) obtained from the umbilical arteries reflect downstream blood flow impedance, thus giving indirect evidence of vascular villous tree characteristics. Pulsatility Index, which quantifies FVW's, decreases throughout normal pregnancy, indicating decreasing impedance and is often higher in cases of fetal growth restriction (FGR). Different approaches (morphometrical, morphological, mathematical, immunohistochemical and molecular) have contributed to elucidation of which anomalies of the vascular villous tree underlie Doppler findings. 3D ultrasound may be useful in the study of feto-placental perfusion. However, the unsolved question is why developmental villous tree anomalies occur. Crucial to the success of future research is definition of the population studied based on the uniform and correct definition of FGR.