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IGFBP-4 and −5 are expressed in first-trimester villi and differentially regulate the migration of HTR-8/SVneo… Crosley, Erin J; Dunk, Caroline E; Beristain, Alexander G; Christians, Julian K Dec 4, 2014

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RESEARCHuaCrosley et al. Reproductive Biology and Endocrinology 2014, 12:123http://www.rbej.com/content/12/1/123through proteolysis of the IGFBPs [7]. In addition to1Biological Sciences, Simon Fraser University, V5A 1S6 Burnaby, CanadaFull list of author information is available at the end of the articleBackgroundAdverse gestational outcomes such as preeclampsia (PE)and intrauterine growth restriction (IUGR) are thoughtto be caused, at least in part, by deficiencies in processescritical to placental development, including extravilloustrophoblast (EVT) invasion. EVT invade the maternaldecidua and replace the endothelium of uterine spiralarteries, thus increasing vessel diameter to ensure adequateblood flow required for oxygen and nutrient delivery to theplacenta [1,2]. In both human villous explants and primarytrophoblast cultures, insulin-like growth factors I and II(IGF-I and IGF-II, respectively), stimulate trophoblast pro-liferation and EVT migration [3,4].The bioavailability of IGF-I and -II is modulated by sixinsulin-like growth factor-binding proteins (IGFBPs)[3,5,6], with the release of the IGFs generally achieved* Correspondence: julian_christians@sfu.caTrophoblast migrationAbstractBackground: Adverse gestational outcomes such as preeclampsia (PE) and intrauterine growth restriction (IUGR)are associated with placental insufficiency. Normal placental development relies on the insulin-like growth factors -Iand -II (IGF-I and -II), in part to stimulate trophoblast proliferation and extravillous trophoblast (EVT) migration. Theinsulin-like growth factor binding proteins (IGFBPs) modulate the bioavailability of IGFs in various ways, includingsequestration, potentiation, and/or increase in half-life. The roles of IGFBP-4 and −5 in the placenta are unknown, despiteconsistent associations between pregnancy complications and the levels of two IGFBP-4 and/or −5 proteases,pregnancy-associated plasma protein -A and -A2 (PAPP-A and PAPP-A2). The primary objective of this study wasto elucidate the effects of IGFBP-4 and −5 on IGF-I and IGF-II in a model of EVT migration. A related objective wasto determine the timing and location of IGFBP-4 and −5 expression in the placental villi.Methods: We used wound healing assays to examine the effects of IGFBP-4 and −5 on the migration of HTR-8/SVneocells following 4 hours of serum starvation and 24 hours of treatment. Localization of IGFBP-4, −5 and PAPP-A2 wasassessed by immunohistochemical staining of first trimester placental sections.Results: 2 nM IGF-I and -II each increased HTR-8/SVneo cell migration with IGF-I increasing migration significantly morethan IGF-II. IGFBP-4 and −5 showed different levels of inhibition against IGF-I. 20 nM IGFBP-4 completely blockedthe effects of 2 nM IGF-I, while 20 nM IGFBP-5 significantly reduced the effects of 2 nM IGF-I, but not to controllevels. Either 20 nM IGFBP-4 or 20 nM IGFBP-5 completely blocked the effects of 2 nM IGF-II. Immunohistochemistryrevealed co-localization of IGFBP-4, IGFBP-5 and PAPP-A2 in the syncytiotrophoblast layer of first trimester placentalvilli as early as 5 weeks of gestational age.Conclusions: IGFBP-4 and −5 show different levels of inhibition on the migration-stimulating effects of IGF-I andIGF-II, suggesting different roles for PAPP-A and PAPP-A2. Moreover, co-localization of the pappalysins and theirsubstrates within placental villi suggests undescribed roles of these molecules in early placental development.Keywords: Pappalysins, PAPP-A, PAPP-A2, Insulin-like growth factor-binding proteins, IGFBP-4, IGFBP-5,IGFBP-4 and −5 are exprevilli and differentially regHTR-8/SVneo cellsErin J Crosley1, Caroline E Dunk2, Alexander G Beristain3,4© 2014 Crosley et al.; licensee BioMed CentralCommons Attribution License (http://creativecreproduction in any medium, provided the orDedication waiver (http://creativecommons.orunless otherwise stated.Open Accessssed in first-trimesterlate the migration ofnd Julian K Christians1*Ltd. This is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andiginal work is properly credited. The Creative Commons Public Domaing/publicdomain/zero/1.0/) applies to the data made available in this article,Crosley et al. Reproductive Biology and Endocrinology 2014, 12:123 Page 2 of 7http://www.rbej.com/content/12/1/123reducing the availability of the IGFs, in some contextsIGFBPs increase the half-life of IGFs, concentrate themin particular regions and/or potentiate their effects [8].Furthermore, IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-5all exert IGF-independent effects in a variety of cellmodels and tissues [3,6,9]. For example, IGFBP-1 stimu-lates migration in an IGF-independent manner in HTR-8/SVneo cells, an immortalized trophoblast cell line com-monly used to model EVT migration and invasion [10]. InBeWo cells, an immortalized choriocarcinoma cell linecommonly used to model the villous cytotrophoblast,IGFBP-3 inhibits proliferation in an IGF-independentmanner [11]. The proteolytic fragments of cleaved IGFBP-3 and −5 have also been shown to have effects in othersystems [12-14].The pappalysins, pregnancy-associated plasma proteins-Aand -A2 (PAPP-A and PAPP-A2, respectively), are twoIGFBP proteases that have been consistently associatedwith a range of pregnancy complications. PAPP-A is aprotease of IGFBP-4 and −5 that is produced by theplacenta [15] and circulates in the maternal blood athigh levels during pregnancy. Abnormally low levels ofPAPP-A in the first trimester have frequently been associ-ated with increased risk of PE and IUGR [16]. Pregnancy-associated plasma protein-A2 (PAPP-A2) shares 45%amino acid identity with PAPP-A, cleaves IGFBP-5 but notIGFBP-4, and is also produced by the syncytiotrophoblastand released into the maternal circulation during preg-nancy [17]. PAPP-A2 levels in the placenta and maternalcirculation are higher at term in preeclamptic pregnan-cies and pregnancies with severe fetal growth restriction[18-21], and PAPP-A2 levels are also elevated in thefirst-trimester maternal serum of pregnancies that subse-quently develop preeclampsia [22].The mechanistic links between circulating pappalysinlevels and pregnancy complications, and whether alteredpappalysin expression plays a causal role in placentalpathologies, remain unknown. However, whatever rolethe pappalysins play in placental development and physi-ology likely involves their IGFBP substrates, IGFBP-4and −5. While IGFBP-1 is the most abundant IGFBPwithin the maternal decidua [23] and is relatively well-studied, the roles of IGFBP-4 and −5 in placental devel-opment have received little attention.The purpose of this study was to investigate whetherthe pappalysins might influence EVT migration and inva-sion through effects on IGF availability and/or other ac-tions of the IGFBPs. We employed a well-establishedmodel of first trimester EVT to examine the effects of ex-ogenous IGFBP-4 and −5 on the migration-stimulating ef-fects of IGF-I and IGF-II. We focused primarily on theinhibition of the migration, but also tested for potentiationand IGF-independent effects. The IGFBPs are known tobe expressed in the maternal decidua [23] and thereforeare well positioned to regulate EVT invasion. However, toexamine whether IGFBP-4 and −5 might also regulateprocesses within the villi, we examined the expressionof these binding proteins in first-trimester villi usingimmunohistochemistry.MethodsCell lines and cell cultureHTR-8/SVneo cells, an immortalized cell line that is awell-established model of first trimester human tropho-blasts, were obtained from Dr. Charles Graham (Queen’sUniversity, Kingston, ON, Canada) [24,25]. Cells werecultured in RPMI 1640 medium supplemented with 10%fetal bovine serum, 100 U/mL of penicillin, and 100 U/mLof streptomycin at 37°C in a humidified atmosphere of 5%CO2. Cell culture media and reagents were purchased fromLife Technologies (Burlington, ON, Canada).HTR-8/SVneo cell wounding assayHTR-8/SVneo cells were seeded at a density of 40,000cells per well in 24-well polystyrene tissue culture platesand allowed to grow to confluence before serum starvingfor at least 4 hours. Wounding was performed with a20 μL pipette tip across the horizontal midsection ofeach well, and photographs were taken at two pointsalong each wound. The XY coordinates of each pointwere saved using Simple PCI coordinate mapping soft-ware and photographed again 24 hours after treatment(described below). The percentage wound closure wascalculated as (1 – (area of wound at 24 hours/area ofwound at time 0)) ×100%, as quantified using ImageJsoftware (see Additional file 1: Figure S1). Recombinanthuman IGF-I, IGF-II, IGFBP-4 and IGFBP-5 were pur-chased from R&D Systems (Minneapolis, Minnesota). Adose response experiment ranging from 2–25 nM IGF-Ior –II established that 2 nM was sufficient to generate asignificant increase in migration of HTR-8/SVneo cells.Dosage in the range of 1–25 nM IGFBP-4 or −5 was ex-pected to have inhibitory effects based on the literature[26] and preliminary experiments. Migration experimentsexamining the stimulatory effects of the IGFs were per-formed by treating HTR-8/SVneo cells for 24 hours witheither 2 nM IGF-I or IGF-II. Migration experimentsexamining the inhibitory effects of the IGFBPs were per-formed by co-treating HTR-8/SVneo cells with either20 nM IGFBP-4 or IGFBP-5, and one of the twoIGFs at 2 nM. Experiments examining IGF-I potentiatingeffects of low doses of IGFBP-5 were performed across arange of concentrations from equimolar amounts of IGF-Iand IGFBP-5, to an 8 fold excess of IGF-I against IGFBP-5based on previous work with IGFBP-1 [27,28]. Potentiatingeffects of IGFBP-4 were not investigated because we knowof no evidence that IGFBP-4 has IGF-potentiating effectsin other systems [29]. Preliminary experiments examiningan intermediate level (56%). Either 20 nM IGFBP-4or 20 nM IGFBP-5 completely blocked the effects of2 nM IGF-II (Figure 2). The migration-stimulating effectsabab bbb b0 nM 2 nM 10 nM 25 nM 0 nM 2 nM 10 nM 25 nMWound closure (%)020406080100Figure 1 Effects of IGF-I and IGF-II dose on migration of HTR-8/SVneo cells. IGF-I and –II experiments were conducted separatelyand therefore have different control values. Values are mean ± standarderror of the mean, and values with different superscript letterswithin an experiment are significantly different according to Tukey’sHSD test (overall variation in IGF-I experiments: F3, 20 = 28.65, P < 0.0001,acabaabWound closure (%)0102030405060702 nM IGF-I - + + + - - -2 nM IGF-II - - - - + + +20 nM IGFBP-4 - - + - - + -20 nM IGFBP-5 - - - + - - +Figure 2 Effects of IGF-I, IGF-II, IGFBP-4 and IGFBP-5 on migrationof HTR-8/SVneo cells. Values are mean ± standard error of the mean,and values with different superscript letters are significantly differentCrosley et al. Reproductive Biology and Endocrinology 2014, 12:123 Page 3 of 7http://www.rbej.com/content/12/1/123potential IGF-independent effects of 20 nM IGFBP-4 or20 nM IGFBP-5 were also performed.ImmunohistochemistryHuman placental tissue was collected from elective termi-nations of pregnancies between 5 and 13 weeks of gesta-tion. Sections from a total of 6 different placentae wereused. Upon collection, samples were fixed overnight in 4%paraformaldehyde, paraffin-embedded, sectioned (4–5 μm)and mounted. Sections were deparaffinized in xylene andrehydrated with a graded series of alcohol. Antigen re-trieval was performed by heating sections in 10 mM citratebuffer for 30 min. Immunohistochemical staining wasperformed using a horseradish peroxidase- 3-amino-9-ethylcarbazole (HRP-AEC) goat kit (R&D Systems)according to the manufacturer’s instructions. Adjacentsections were incubated with primary polyclonal anti-human antibodies against one of cluster of differentiation31 (CD31), PAPP-A2, IGFBP-4 or IGFBP-5, all of whichwere raised in goat (R&D Systems). Immunoreactivity wasvisualized with AEC and sections were counterstainedwith hematoxylin. Negative controls were prepared usingnon-specific goat immunoglobulin G (IgG; R&D Systems)in place of primary antibodies. The use of these sam-ples was approved by the University of British ColumbiaChildren’s and Women’s Research Ethics Board, the MountSinai Hospital Research Ethics Board and the Simon FraserUniversity Research Ethics Board.Statistical analysesEffects of treatments were analyzed in JMP (ver. 10; SASInstitute Inc.) using a general linear model including theeffects of treatment and date of experiment. Treatmentreplicates were distributed across different experimentaldates, with replication both within and between days. Allexperiments contained the appropriate controls (controls,and IGF-I and/or –II alone) to allow for comparison acrossdates (with similar sample sizes for treatments performedon the same day).ResultsIGF-I, IGF-II and HTR-8/SVneo migrationPreliminary experiments with 2 nM, 10 nM or 25 nM ofeither IGF-I or IGF-II showed a significant stimulatory ef-fect of 2 nM on HTR-8/SVneo cells (Figure 1). TreatingHTR-8/SVneo cells with either IGF-I or IGF-II at 2 nMsignificantly increased migration compared with controls,with IGF-I increasing migration significantly more thanIGF-II (Figure 2).IGFBP-4, IGFBP-5 inhibit effects of IGF-I and IGF-II onHTR-8/SVneo migrationWhile IGFBP-4 and −5 both significantly reduced the ef-fects of 2 nM IGF-I, 20 nM IGFBP-4 almost completelyblocked the effects of 2 nM IGF-I, whereas 20 nMIGFBP-5 did not (Figure 2). While 2 nM IGF-I in-creased wound closure to 63%, co-treatment with 20 nMIGFBP-4 reduced wound closure to 41% compared with38% in controls, but co-treatment with 20nM IGFBP-5 hada lesser effect, reducing IGF-I-mediated wound closure toN = 6 per treatment; IGF-II experiments: F3, 43 = 12.86, P < 0.0001,N = 6–14 per treatment).according to Tukey’s HSD test (overall variation among treatments:F6, 249 = 38.11, P < 0.0001, N = 24–62 per treatment).of IGF-II (wound closure 53%) were completely blocked by20 nM IGFBP-4 (wound closure 36%) or 20 nM IGFBP-5(wound closure 42%); neither IGFBP treatment was signifi-cantly different from controls (38%). There was no signifi-cant effect of IGFBP-4 or IGFBP-5 alone at 20 nM inpreliminary experiments, suggesting an absence of IGF-independent effects (data not shown). Moreover, there wasno significant potentiation effect of low doses of IGFBP-5with 2 nM IGF-I (Figure 3).Immunoreactivity for IGFBP-4, IGFBP-5 and PAPP-A2 inthe syncytiotrophoblastThe presence of IGFBP-4, IGFBP-5 and PAPP-A2 wasexamined in serial sections of placental villi at 5, 6, 7and 13 weeks of gestation. In addition to the non-specific goat IgG negative control, staining against CD31was used as a negative control for immunoreactivity inthe syncytiotrophoblast, since staining was only expectedCrosley et al. Reproductive Biology and Endocrinology 2014, 12:123 Page 4 of 7http://www.rbej.com/content/12/1/123in the endothelium of fetal vessels. Across all gestationalages, IGFBP-4 and −5 showed localization within thesyncytiotrophoblast, with stronger immunoreactivity ob-served for IGFBP-4 than IGFBP-5 (Figure 4). IGFBP-4and −5 also appeared in the chorionic mesoderm. PAPP-A2 in contrast, showed little to no immunoreactivityin the chorionic mesoderm at 5 and 6 weeks, but didshow strong immunoreactivity in the syncytiotrophoblast(Figure 4).DiscussionTo investigate the potential mechanism underlying theassociations between levels of PAPP-A and PAPP-A2and pregnancy complications such as PE and IUGR,we studied the effects of pappalysin substrates IGFBP-4ab b ba,b+ 2 nM IGF-IIGFBP-5: 0 nM 0 nM 0.25 nM 0.5 nM 2 nMWound closure (%)020406080Figure 3 Effects of low doses of IGFBP-5 on migration ofHTR-8/SVneo cells. Values are mean ± standard error of the mean,and values with different superscript letters are significantly differentaccording to Tukey’s HSD test (overall variation among treatments:F4, 42 = 4.38, P < 0.0048, N = 6–14 per treatment).and −5 on IGF-I and -II in a model of EVT migration.We also examined the location and timing of IGFBP-4and −5 expression to determine whether the pappalysinsmight also influence processes occurring within the villiearly in pregnancy.Consistent with previous findings in various modelsof first trimester human EVT [30-36], both IGF-I andIGF-II at 2 nM significantly stimulated migration ofHTR-8/SVneo cells in a cell-wounding assay. Further-more, IGF-I had greater stimulatory effects than IGF-IIat this concentration. The difference in effects of IGF-Iand –II may be due to the presence of both type 1 andtype 2 IGF receptors (IGF1R and IGF2R) in HTR-8/SVneo cells [26,36], and the higher binding affinity ofIGF1R for IGF-I than for IGF-II [37].IGFBP-4 and −5 showed different levels of IGF inhib-ition. IGFBP-4 was able to block the migration-stimulatingeffects of both IGF-I and IGF-II to control levels. Incontrast, IGFBP-5 was able to inhibit the migration-stimulating effects of IGF-II to control levels, but only par-tially inhibited IGF-I. While previous work has shownIGFBP-5 blocks IGF-II stimulation of migration in a cellline model of EVT [26], this is to our knowledge the firstdata regarding the effects of exogenous IGFBP-4 ontrophoblast migration. IGFBP-4 has however been foundto influence migration and invasion in cancer studies, withinhibitory or stimulatory effects on migration dependingon the model examined [38-42]. Similarly, the effects ofIGFBP-5 on cellular proliferation and invasion in breastcancer studies appear to be cell line dependent [43].The inhibitory effect of IGFBP-4 on trophoblast mi-gration may, at least in part, underlie the associationbetween elevated circulating levels of IGFBP-4 in earlypregnancy and the subsequent development of fetal growthrestriction [44].There was no evidence of potentiation of the effectsof IGF-I by low doses of IGFBP-5, either because nosuch effect exists in vivo, or because IGF potentiation re-quires interaction between IGFBP-5 and the extracellularmatrix [45]. Similarly, the lack of IGF-independent ef-fects of IGFBP-4 or −5 in our experiments may be dueto a real absence of effects in vivo, to the absence of as-yet-uncharacterized cell surface IGFBP-4 and −5 recep-tors in HTR-8/SVneo cells [29], or to a requirement forinteraction with the extracellular matrix [45] or prote-olysis of the IGFBPs [12,14].IGF-I and -II have different primary sources in the pla-centa. IGF-II is strongly expressed by EVT and syncytio-trophoblast in the human placenta [3,4,23], whereas thepredominant source of IGF-I in the placenta is the ma-ternal circulation, as it is only weakly expressed by theplacenta [4,23]. Low levels of IGF-I mRNA relative toIGF-II mRNA have been detected in the cytotrophoblast,mesodermal core and endothelium of human placentalCrosley et al. Reproductive Biology and Endocrinology 2014, 12:123 Page 5 of 7http://www.rbej.com/content/12/1/123Negative IGFBP-45 week 5 week enlarged eek villi (in first, second and third trimesters), but not inEVT [30,46,47]. IGF-I has been detected less consistentlyat the protein level. In one study, IGF-I protein wasundetectable in human placental lysates [48], and inanother IGF-I protein was detected in first trimestervillous cytotrophoblasts, but at much lower levels thanIGF-II [47].A number of observations from previous work andthis study suggest that PAPP-A and PAPP-A2 play verydifferent roles in normal placental development and dis-ease, which may explain why PAPP-A and PAPP-A2 showcontrasting levels in relation to adverse pregnancy out-comes (i.e., PAPP-A being down-regulated and PAPP-A2being upregulated in association with pregnancy complica-tions [16]). PAPP-A is a protease of both IGFBP-4 andIGFBP-5, whereas PAPP-A2 only proteolyzes IGFBP-5.Therefore our data suggest that PAPP-A2 has less of astimulatory effect on EVT migration than PAPP-A, asIGFBP-5 shows less inhibition of IGF-I, which is morepotent than IGF-II. Furthermore, PAPP-A may have a6 w6 week enlarged Figure 4 Immunoreactivity (red colour) against IGFBP-4, −5, and PAPPSerial cross sections of placental villi at 5 and 6 weeks of gestation (7 and 1Negative controls used non-specific goat IgG in the place of the primaST = syncytiotrophoblast. Scale bars denote 100 μm.IGFBP-5 PAPP-A2stronger effect modulating the availability of IGF-I fromthe maternal circulation than PAPP-A2.PAPP-A and PAPP-A2 are expressed by the syncytio-trophoblast as well as invasive EVT [49,50]. It was previ-ously thought that the only location of expression ofIGFBP-4 and −5 in the first trimester was the maternaldecidua, since IGFBP-5 had only been observed in villiin the second and third trimester [23]. More recently,IGFBP-4 immunoreactivity has been observed in thechorionic mesoderm of placental villi at 10–13 weeks[44]. Here, we show that IGFBP-4 and −5 are present inthe syncytiotrophoblast of placental villi as early as5 weeks of gestation. PAPP-A and PAPP-A2 may there-fore influence aspects of early placental development inaddition to the EVT invasion of the decidua and remod-eling of the spiral arteries, e.g., cytotrophoblast prolifera-tion or fusion with the syncytiotrophoblast. While we donot know whether the placentae we sampled were frompregnancies that would have gone on to develop preg-nancy complications, we observed consistent results inA2 in the syncytiotrophoblast of first trimester placental villi.3 weeks not shown) were stained for IGFBP-4, IGFBP-5, and PAPP-A2.ry antibodies. FB = fetal blood vessel, CM = chorionic mesoderm,Crosley et al. Reproductive Biology and Endocrinology 2014, 12:123 Page 6 of 7http://www.rbej.com/content/12/1/1236 different placentae, 3 of which were at a gestationalage of 7 weeks or less. It is very unlikely that none ofthese early placentae were from a healthy, uncompli-cated pregnancy.ConclusionsIGFBP-4 and −5 have differing effects on IGF-I and -IIin an in vitro model of EVT migration. Given thatPAPP-A and PAPP-A2 show contrasting patterns ofregulation in adverse pregnancy outcomes, we propose thatthe stimulatory effects of PAPP-A2 on EVT migration maybe less than those of PAPP-A, and that PAPP-A2 mayfunction principally to modulate IGF-II produced locally inthe placenta via its effects on IGFBP-5. Moreover, giventhe lack of IGF-independent and potentiating effects of thepappalysin substrates, we propose that the effects of thepappalysins are principally stimulatory on migration. Thishypothesis suggests that low PAPP-A levels in early preg-nancy may contribute to placental pathology, whereas highPAPP-A2 levels in pregnancies that go on to develop com-plications may represent a compensatory response [51,52].Additional fileAdditional file 1: Figure S1. HTR-8/SVneo cell wounding assay.AbbreviationsAEC: 3-amino-9-ethylcarbazole; CD31: Cluster of differentiation 31;EVT: Extravillous trophoblast; HRP: Horseradish peroxidase; IGF: Insulin-likegrowth factor; IGF1R: Insulin-like growth factor 1 receptor; IGF2R: Insulin-likegrowth factor 2 receptor; IGFBP: Insulin-like growth factor bindingprotein; IgG: Immunoglobulin G; IUGR: Intrauterine growth restriction;PAPP-A: Pregnancy-associated plasma protein–A; PE: Preeclampsia.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsEJC performed all cell culture work and statistical analyses and drafted themanuscript. CED and AGB provided guidance with methodology, preparedsections for immunohistochemistry, and provided comments on themanuscript. JKC conceived of the study, participated in its design, performedthe immunohistochemistry, and helped to draft the manuscript. All authorsread and approved the final manuscript.AcknowledgementsThis work was supported by a Canadian Institutes of Health ResearchMaster’s Award (Frederick Banting and Charles Best Canada GraduateScholarships), Simon Fraser University (SFU) graduate fellowships and aPhyllis Carter Burr Scholarship to EJC, and SFU Vice-President, Research,Bridging and NSERC Discovery Grants to JKC. We thank Dr. Gordon Rintoulfor the use of his cell culture facilities at Simon Fraser University, Pavel Ogayfor laboratory assistance, and Bryce Pasqualotto, Lubna Nadeem, Bo Pengand Tim Beischlag for advice and guidance.This paper is dedicated to the memory of Dr. Andrée Gruslin, who providedguidance and mentorship in this and so much of our other work.Author details1Biological Sciences, Simon Fraser University, V5A 1S6 Burnaby, Canada.2Research Centre for Women’s and Infants Health, Lunenfeld Tanenbaum3Research Institute, Mount Sinai Hospital, Toronto, Canada. Department ofObstetrics and Gynecology, The University of British Columbia, Vancouver,Canada. 4The Child and Family Research Institute, Vancouver, Canada.Received: 8 October 2014 Accepted: 26 November 2014Published: 4 December 2014References1. Knofler M: Critical growth factors and signalling pathways controllinghuman trophoblast invasion. Int J Dev Biol 2010, 54:269–280.2. Redman CW, Sargent IL: Latest advances in understanding preeclampsia.Science 2005, 308:1592–1594.3. Nayak NR, Giudice LC: Comparative biology of the IGF system inendometrium, decidua, and placenta, and clinical implications for foetalgrowth and implantation disorders. Placenta 2003, 24:281–296.4. Sferruzzi-Perri AN, Owens JA, Pringle KG, Roberts CT: The neglected role ofinsulin-like growth factors in the maternal circulation regulating fetalgrowth. J Physiol 2011, 589:7–20.5. Murphy LJ: Insulin-like growth factor-binding proteins: functional diversity orredundancy? J Mol Endocrinol 1998, 21:97–107.6. 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