ARTICLE IN PRESS

Acta histochemica 112 (2010) 203—214

0065-1281/$ - sdoi:10.1016/j.

�CorrespondE-mail addr

www.elsevier.de/acthis

REVIEW

Vasculogenesis and angiogenesis in theendometrium during menstrual cycle andimplantation

Ramazan Demira,�, Aylin Yabaa, Berthold Huppertzb

aDepartment of Histology and Embryology, Faculty of Medicine, Akdeniz University, 07070 Antalya, TurkeybInstitute of Cell Biology, Histology and Embryology, Center for Molecular Medicine, Medical University of Graz, Austria

Received 23 March 2009; received in revised form 26 March 2009; accepted 4 April 2009

KEYWORDSVasculogenesis;Angiogenesis;Implantation;Review

ee front matter & 2009acthis.2009.04.004

ing author. Tel.: +90 242ess: rdemir@akdeniz.ed

SummaryBlood vessels develop via two subsequent processes, vasculogenesis and angiogen-esis, both being of crucial importance during menstrual cycle and implantation.These processes are also involved in the development of the fetal and placentalvasculatures. During vasculogenesis, formation of the earliest primitive capillaries isachieved by in situ differentiation of hemangiogenic stem cells that are derived frompluripotent mesenchymal cells. The subsequent process, angiogenesis, is character-ized by development of new vessels from already existing vessels, and is a wellcoordinated process initiated by stimulation of various growth factors. Vasculogen-esis and angiogenesis are important and complex processes involving extensiveinterplay between cells and growth factors. The development, maturation andmaintenance of the vascular network are necessary for successful hemochorialplacentation as well as normal embryonic development and growth. In this review,we outline the basic mechanisms of vasculogenesis and angiogenesis in theendometrium during the menstrual cycle and different stages of implantation, andconsider how this data can be applied to human pregnancy. Recent studies haveshown that during the initiation steps of implantation, angiogenic factors triggervasculogenesis and angiogenesis. Different inducers and stimulators affect angio-genesis and vasculogenesis by directly or indirectly stimulating proliferation,differentiation and migration of endothelial or respective precursor cells. As aconclusion, understanding the mechanisms of angiogenesis and the roles ofangiogenic factors during the menstrual cycle and implantation may provide newinsights and possible approaches for embryo implantation and healthy pregnancy.& 2009 Elsevier GmbH. All rights reserved.

Elsevier GmbH. All rights reserved.

2496881; fax: +90 242 227 44 86.u.tr (R. Demir).

ARTICLE IN PRESS

R. Demir et al.204

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204Mechanisms of angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205

Sprouting angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205Intussusception . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205Vessel elongation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205Incorporation of circulating endothelial precursor cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205

Molecules promoting angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206Vascular endothelial growth factor (VEGF) and its receptors . . . . . . . . . . . . . . . . . . . . . . . . . . 206Fibroblast growth factor (FGF) and its receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206Epidermal growth factor (EGF) and its receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207Neuropilin (NP) -1 and -2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207Angiopoietins (Ang) and their receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207

Angiogenesis during the menstrual cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207Angiogenesis during post-implantation periods of the endometrium . . . . . . . . . . . . . . . . . . . . . . . . . . 208Hormonal control of endometrial angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

Human chorionic gonadotrophin (hCG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209Estrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209Progesterone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210

Conclusions and prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

Introduction

Blood vessel development comprises variousmechanisms subsumed in two distinct processes:vasculogenesis and angiogenesis.

Vasculogenesis is the formation of first bloodvessels through differentiation and migration ofpluripotent mesenchymal cells (hemangiogenic pro-genitor cells). These cells differentiate into hemato-poietic progenitor cells to generate blood cells andinto angioblastic progenitor cells to generate bloodvessels. However, so far it has not been establishedwhether angioblastic progenitor cells and angioblastsare the same cells. There is evidence that duringvasculogenesis and angiogenesis these cells can begenerated from each other (Demir et al., 2007).

Vasculogenesis mainly occurs during embryonicand fetal development, although recruitment ofangioblasts from bone marrow and peripheral bloodin response to an ischemic insult has beendescribed in adults as well (Zygmunt et al., 2003).

Vasculogenesis comprises three important steps:(1) stimulation of pluripotent mesenchymal stemcells followed by (2) their proliferation anddifferentiation into hemangiogenic progenitor cellsand (3) derivation of angioblastic and hematopoie-tic cell populations (Demir et al., 2006, 2007).

Single endothelial vessels developed duringvasculogenesis are used as templates for angiogenicprocesses to develop a vascular network.

Angiogenesis is the development of new micro-vessels from already existing blood vessels. Thus,

angioblasts do not have a role in this process;rather proliferative endothelial cells develop newvascular structures (Huppertz and Peeters, 2005).Also circulating endothelial progenitor cells (EPCs)may be involved in angiogenesis. Due to individualfactors in the systemic circulation, the number ofdifferentiating circulating EPCs can be altered. Itwas suggested that an inadequate vascular devel-opment could be associated with a reducednumbers of circulating EPCs (Lambiase et al.,2004).

Angiogenesis normally occurs during embryonicand fetal growth and development, and only rarelyoccurs in the adult. Such exceptions are woundhealing, in specific diseases (e.g., diabetic retino-pathy, tumor growth) and during the menstrualcycle (Torry and Torry, 1997; Augustin, 2000; Arroyoand Winn, 2008).

In most organs, there is no need to form newblood vessels unless they are injured by trauma orinfection. Physiologic angiogenesis only plays animportant role in wound and fracture healing,formation of corpus luteum, endometrial growth,embryo implantation and placentation. Commonly,the angiogenic process is initiated by growthfactors such as basic fibroblast growth factor(bFGF), vascular endothelial growth factor (VEGF),placental growth factor (PlGF), and tumor necrosisfactor alpha (TNFa). The effects of these growthfactors include: increasing the permeability ofvessels (Folkman and Klagsbrun, 1987), stimulating

ARTICLE IN PRESS

Vasculogenesis and angiogenesis during menstrual cycle and implantation 205

proteolytic degradation of the extracellular matrix(basal and reticular lamina) activating specificproteases (collagenases and plasminogen activa-tors) and causing proliferation of endothelial cells.Proliferation of endothelial cells is followedby migration and invasion of these cells towardsthe extracellular matrix. A vessel lumen is formedthrough intra- or extracellular mechanisms, eitherfrom intracellular vacuoles or extracellular chan-nels. Finally, the functional maturation of theendothelium (recruitment of pericytes and smoothmuscle cells) concludes angiogenesis (Reynoldset al., 1992; Risau, 1997; Zygmunt et al., 2003).

The endometrium is one of the few tissues in theadult where physiological angiogenesis normallyoccurs. Studies of endometrial angiogenesis arecomplicated by (1) the continual changes due totissue growth and regression during the menstrualcycle, as well as (2) the differences between thetwo different layers of the endometrium: functio-nalis and basalis (Weston and Rogers, 2000).

Mechanisms of angiogenesis

Angiogenesis is a complex process where variousfactors have important roles. Vascular sprouting isthe basic start of this process and depends onenvironmental conditions such as: (1) presence ofangiogenic factors, (2) presence of hypoxia tostimulate growth, (3) secretion of essential pro-teases by endothelial cells for destruction of tissues,(4) movement/migration ability of endothelial cells,and (5) an appropriate proliferation rate of en-dothelial cells to secure outgrowth of a new vessel.

So far, four different angiogenic mechanismshave been detected which lead to the formationof new blood vessels: sprouting angiogenesis,intussusception, elongation/widening, and incor-poration of circulating endothelial precursor cellsinto vessel walls.

Sprouting angiogenesis

For a long time this was the only knownmechanism. It occurs during neovascularization ofavascular tissues, such as the rapidly growingcorpus luteum, or vascular invasion of growingtumors (Girling and Rogers, 2005). It includesendothelial cell activation, basement membranedegradation, migration of endothelial cells, theirproliferation, tube formation, stabilization withpericyte recruitment and extracellular matrixreformation.

Vasodilator-stimulated phosphoprotein (VASP)may participate in endothelial sprouting during

vasculogenesis, and one of the effects of vascularendothelial growth factor and interleukin-8 (IL-8)in angiogenesis may be to induce VASP expression ina paracrine manner (Kayisli et al., 2002). It hasbeen suggested that sprouting of new vessels fromrat aorta sections grown in collagen depends on theproduction of pro-angiogenic cytokines such asVEGF and IL-8 (D’Alessandro et al., 2007). Inaddition, our recent results suggest that apoptosisis likely to be involved in the physiologic mechan-isms of placental vasculogenesis and angiogenesis,such as lumen formation and angiogenic branching(Tertemiz et al., 2005; Seval et al., 2007).

Intussusception

The second mechanism of angiogenesis, intussus-ception, is the internal division or splitting of a vesselin two by the deposition of material in the center ofthe vessel lumen. This is an alternative mechanismto the sprouting mode of angiogenesis. The intussus-ceptive microvascular growth divides the existingvessel lumen by formation and insertion of tissuefolds and columns of interstitial tissue into the vessellumen. The tissues developing during intussusceptionare also termed interstitial or intervascular tissuestructures and tissue pillars or posts. Intussusceptionalso includes the establishment of new vessels by insitu loop formation in the wall of large veins (Patan,2004). It has been suggested that intussusceptionshave been implicated in three processes of vasculargrowth and remodeling: (1) intussusceptions permitsrapid expansion of the capillary plexus, furnishinga large endothelial surface for metabolic exchange;(2) arborizations cause changes in the size, positionand form of preferentially perfused capillary seg-ments, creating a hierarchical tree; and (3) branch-ing remodeling leads to modification of the bran-ching geometry of supplying vessels, optimizing pre-and post-capillary flow properties (Djonov et al.,2003).

Vessel elongation

Vessel elongation/widening is the third mechan-ism and, like intussusception, the vessel wall is notbreached during this process.

Incorporation of circulating endothelialprecursor cells

A possible fourth mechanism of angiogenesisis the incorporation of circulating endothelialprecursor cells from the bone marrow into bloodvessels. This mechanism of angiogenesis was also

ARTICLE IN PRESS

R. Demir et al.206

called ‘‘postnatal vasculogenesis’’ (Weston andRogers, 2000). Incorporation of circulating en-dothelial precursor cells seems to play an impor-tant role during pregnancy. Pregnant womensuffering from preeclampsia show decreased num-bers and senescence of endothelial progenitor cells(Sugawara et al., 2005).

Molecules promoting angiogenesis

The angiogenic process is initiated by stimulationof growth factors. Some of the angiogenic factorspositively promote angiogenesis, while others showa negative effect. Thus, angiogenesis occurs as aresult of a well coordinated balance of factors.Growth factors positively promoting the angiogenicprocess include vascular endothelial growth factorVEGF (VEGF-A, -B, -C, -D, -E) and its receptors(VEGFR-1, -2, -3); PlGF; epidermal growth factor(EGF) and its receptors; FGF and its receptors; NP-1and -2; and the angiopoietin/tie systems (Zygmuntet al., 2003; Demir et al., 2004, 2006; Kayisli et al.,2006; Seval et al., 2008).

Expression of growth factors promoting vascularregeneration such as transforming growth factoralpha (TGF-a), EGF, and VEGF was increased intransplanted organs (Ueno et al., 2006). In general,peptide growth factors signaling through cognatereceptors with tyrosine kinase intracellular signal-ing domains such as FGFR, EGFR, IGFR, PDGFR andc-met are known to stimulate embryonic morpho-genesis (Warburton et al., 2000).

Vascular endothelial growth factor (VEGF)and its receptors

The VEGF family of proteins is one of the crucialregulators of angiogenesis, lymphangiogenesis andvasculogenesis. VEGF is an endothelial cell mitogenand angiogenic inducer produced by a variety ofcells and tissues. It exhibits a specific affinity forvascular endothelial cells, and thus VEGF has thepotential to play an important role in the develop-ment of the vascular system (Jakeman et al., 1993;Huppertz et al., 2007). The VEGF family consists ofsix members: VEGF-A, -B, -C, -D, -E and placentalgrowth factor (PlGF). These ligands bind to threereceptors: VEGFR-1, -2 and -3 (Smith, 2001). Thedifferent isoforms appear to have similar functions,but differ in their binding affinities to theirreceptors as well as to the extracellular matrix.The predominant isoforms in the endometrium aresplice variants of VEGF-A: VEGF121 and VEGF165(Sugino et al., 2002).

PlGF homodimers bind VEGFR-1 and NP-1, whilePlGF heterodimerization with VEGF may also occur.Activation of VEGFR-1 by either PlGF or VEGF-Ainduces different gene expression profiles andphosphorylation of distinct tyrosine residues inthe tyrosine kinase domain of VEGFR-1. Until now,four human isoforms of PlGF (PlGF-1, -2, -3 and -4)have been reported (Shalaby et al., 1995; Chunget al., 2000; Otrock et al., 2007). PlGF null miceare viable and fertile, but they exhibit diminishedvascularization of the retina and the corpusluteum. PlGF enhances VEGF signaling, and PlGFexpression may obviate anti-VEGF based therapy(Carmeliet et al., 2001; Huppertz et al., 2007).

Knockout experiments showed that if mice have adefect in VEGF expression or do not express VEGFreceptors, they are not successful in developing avascular network and thus abort. Hence, the studysuggests that VEGF has an important role inangiogenesis (Evans et al., 1998). In VEGF-knockoutmice (Ferrara et al., 1996; Ferrara, 2000) andVEGFR-2 deficient mice, precursor cells may not beable to differentiate into endothelial cells, which isindispensable for vasculogenesis (Shalaby et al.,1995). On the other hand, a mutation in the locus ofVEGFR-1 (Fong et al., 1995) does not preventdifferentiation of endothelial cells, but ratherresults in endothelial disorganization and abnormalvessel formation during early embryonic develop-ment. During implantation and placentation, de-pending on the needs of increased or decreasedangiogenesis, altered decidual and placental ex-pression of angiogenic factors occurs as a compen-satory mechanism (Plaisier et al., 2007). Theseresults support the idea that VEGF and PlGF andtheir receptors have indeed a very important rolein the normal development of the embryo and itsvasculature.

Fibroblast growth factor (FGF) and itsreceptors

Fibroblast growth factor (FGF) family membersare very effective inducers of endothelial cellmigration, proliferation and tube formation in vitroand are highly angiogenic in vivo (Klagsbrunand D’Amore, 1991). Three members of the FGFfamily, FGF1, 2 and 4, have been detected in thehuman endometrium (Smith, 1998). The epithelialcells showed intense immunopositivity for FGF1and FGF2, while stromal fibroblasts showed lowlevels of labelling. Part of the action of FGFs ininducing angiogenesis is mediated by upregulationof VEGFR-2. Conversely, VEGF promotes angiogen-esis in a synergistic manner with FGF by releasing

ARTICLE IN PRESS

Vasculogenesis and angiogenesis during menstrual cycle and implantation 207

FGF from the extracellular matrix (Smith, 2001).FGF was shown to be upregulated by progesteroneand prolactin in rat endometrium (Zygmunt et al.,2003).

The effects of FGF in different tissues aremediated by the high-affinity tyrosine kinasereceptor FGFR-1, the major FGF receptor in thecardiovascular system. FGF and its receptor play arole in angiogenesis and vascular remodeling (Hyinkand Abrahamson, 1995; Detillieux et al., 2003).

Epidermal growth factor (EGF) and itsreceptors

The expression of EGF and some other oncogeneswas detected in endothelial cells of blood vesselswhich form different vascular structures andcapillary plexuses (Shirayoshi et al., 1997). One ofthe most important oncogenes is TGF-a which isvery important for angiogenesis. EGF is a multi-functional cytokine that also stimulates angiogen-esis and lymphangiogenesis. The EGF receptors arereceptor tyrosine kinases that are activated bybinding their ligands EGF and TGF-a. By activationof the EGF receptors, their ligands can regulateVEGF expression (Pore et al., 2006).

Neuropilin (NP) -1 and -2

Neuropilin NP-1 was identified initially as a 130-to 140-kDa cell-surface glycoprotein that served asa receptor for the semaphorin/collapsins, a largefamily of secreted and transmembrane proteinsthat are used as repulsive guidance signals inaxonal and neuronal outgrowth. NP-1 binds VEGF-A, VEGF-B and PlGF while NP-2 binds VEGF-A,VEGF-C and PlGF. NP-1 acts as a co-receptorenhancing VEGF-A-VEGFR-2 interactions, formingcomplexes with VEGFR-1 and augmenting tumorangiogenesis in vivo (Bagri and Tessier-Lavigne,2002).

Angiopoietins (Ang) and their receptors

The angiopoietin family of proteins collaborateswith the VEGF family to induce growth of bloodvessels during angiogenesis (Suri et al., 1996;Maisonpierre et al., 1997; Valenzuela et al., 1999;Thurston, 2003). So far, four members of theangiopoietin family have been discovered. The fourmembers of this family (Ang-1 to 4) modulateangiogenesis by activating or blocking activation oftheir endothelial receptor Tie-2, a surface receptortyrosine kinase (Suri et al., 1996, Valenzuela et al.,1999; Maisonpierre et al., 1997; Thurston, 2003).

Vascular smooth muscle cells (VSMCs) expressAng-1, which binds to the Tie-2 receptor onendothelial cells to enhance their recruitmentand interaction with perivascular cells and theextracellular matrix. This results in stabilization ofthe endothelial cell–vascular smooth muscle cellinteraction and structural organization of thevessel (Kayisli et al., 2006). Ang-1 induces phos-phorylation of the Tie-2 receptor and preventsapoptosis of endothelial cells. Ang-2 is a naturalantagonist of Ang-1. Although Ang-1 is widelyexpressed in many tissues, Ang-2 is expressedpredominantly at sites of vascular remodeling(Seval et al., 2008), such as those in the femalereproductive tract. Ang-2 also binds to the Tie-2receptor but does not transmit an intracellularsignal. In this situation, the endothelial cellsundergo apoptosis and, in the absence of VEGF,the vessel becomes atrophic (Tertemiz et al.,2005). The biological activity of Ang-2 is modulatedby VEGF. At sites of angiogenic sprouting, VEGF ispresent abundantly. Here, Ang-2 stimulates forma-tion of blood vessels. However, increased Ang-2expression in the presence of low levels of VEGF isassociated with vascular regression (Maisonpierreet al., 1997; Holash et al., 1999).

In the light of the complex changes found at theimplantation site, these mechanisms are of obviousimportance. Ang-1 is found at low levels in stromalfibroblasts of the endometrium and productiondeclines throughout both phases of the menstrualcycle. There is little synthesis of Ang-2 in theproliferative phase of the cycle, although itincreases in the secretory phase (Smith, 2001).

Angiogenesis during the menstrual cycle

As angiogenesis is the formation of new bloodvessels from pre-existing vasculature, it is a processfundamental to the menstrual cycle. The uterineendometrium is a dynamic tissue that undergoesregular cycles of growth and breakdown, and haslong been recognized as one of the few adulttissues where significant angiogenesis occurs on aroutine, physiological basis.

The need for angiogenesis changes spatially andtemporally in the endometrium: during the men-struation phase of the cycle, angiogenesis occurs inthe basal layer, while it occurs in the functional andsub-epithelial capillary plexus during the prolifera-tive and early secretory phases. Throughout thereproductive life of females, angiogenesis regularlyoccurs in the corpus luteum and the endometrium(Gargett and Rogers, 2001) as part of the rapid

ARTICLE IN PRESS

R. Demir et al.208

growth and regression that takes place in thesetissues during the menstrual cycle. There are threedistinct temporal stages when angiogenesis occurs:(1) during menstruation for repair mechanisms ofthe vascular bed, (2) during the proliferative phasefor rapid endometrial growth, and (3) during thesecretory phase when spiral arterioles show sig-nificant growth and coiling (Gargett and Rogers,2001).

Vessel elongation is a major angiogenic mechan-ism in the mid to late proliferative phase ofthe human endometrium (Gambino et al., 2002).Human endometrium synthesizes all members ofthe VEGF-A family, with changing levels dependingon the phase of the cycle. In the proliferative phaseof the cycle, VEGF-A is localized to glandularepithelial and stromal cells. There is no increase inVEGF-A synthesis during the proliferative phase of thecycle. After ovulation, the production of VEGF-A instromal cells declines dramatically and only remainsin surface epithelial cells (Smith, 2001).

In the proliferative phase, angiogenesis occurs inthe functional layer as the endometrium increasesin thickness. In the secretory phase, spiral arter-ioles lengthen and become more coiled, while thesub-epithelial capillary plexus matures. In the post-menstrual phase, angiogenesis is involved in therepair of the superficial layer of the remainingbasal layer of the endometrium. Interestingly,aberrant endometrial angiogenesis resulting inmorphologically abnormal vessels and increasedvascular permeability has been implicated inabnormal uterine bleeding. Based on these find-ings, Nardo (2005) has suggested that circulatinglevels of angiogenic factors fluctuate in a phase-dependent cyclic fashion.

VEGF was shown to increase three-fold in thesecretory phase and further rise towards menstrua-tion as compared to the proliferative phase (Torryet al., 1996). The increase of VEGF in the pre-menstrual period, when ovarian steroids are at lowlevels, could be a prerequisite for menstrualshedding and subsequent tissue regeneration(Perrot-Applanat et al., 2000). The expression ofVEGF and VEGF-Rs during the folliculogenesis/oogenesis in menstrual (ovarian) cycle and fertili-zation processes are illustrated in Figure 1.

Angiogenesis during post-implantationperiods of the endometrium

Angiogenesis is an inevitable step to enableembryo implantation. Various groups have shownthat VEGF and its receptors are significantlyincreased during post-ovulation and around the

peri-implantation period (Gordon et al., 1995; Lichtet al., 2003; Malamitsi-Puchner et al., 2004). Itappears that VEGF expression is highly regulated ina temporal and spatial manner during the earlystages of implantation (Chung et al., 2000; Pooleet al., 2000; Sherer and Abulafia, 2001).

The initiation of angiogenesis begins early duringthe course of implantation and is supported byangiogenic molecules. The decidualized endome-trium is essential for the implantation of thedeveloping embryo and for the maintenance ofpregnancy. Matsui et al. (2004) have demonstratedthat VEGF production increases in association withdecidualization of endometrial stromal cells. Ab-normal expression of VEGF receptors may causelethality during embryogenesis (Fong et al., 1995).In rats, the application of anti-VEGF antibodiessignificantly and dose-dependently lowered embryoimplantation rates (Rabbani and Rogers, 2001;Nardo, 2005).

VEGF promotes uterine vascular permeabilityand dilatation and may also contribute to implan-tation. Just before implantation, and again inearly pregnancy, high levels of VEGFR-1 (Flk-1)are expressed in the pig uterus, in close proximityto endometrial blood vessels (Welter et al.,2003; Postek et al., 2007). VEGF may thuscontribute to the induction of vascular hyperper-meability during implantation and of angiogenesisin the subsequent early pregnancy (Meduriet al., 2000; Kaczmarek et al., 2008). In addition,an increase in the expression of inducible nitricoxide synthase (iNOS) and endothelial nitricoxide synthase (eNOS) at the embryonic sitefollowing implantation may imply a role in vasodi-latation and vascular remodeling (Hickey andFraser, 2000). Thus, VEGF appears to be a keyregulator of angiogenesis and vascular function inthe endometrium. Concentrations of VEGF proteinand VEGF mRNA increase in the secretory and pre-menstrual phases during the menstrual cycle, andcorrelate positively with decidualization (Nardo,2005).

Normal angiogenesis modulated by angiogenicfactors and circulating hormones (steroids andgonadotrophins) plays a critical role in endometrialdevelopment and differentiation during thenormal menstrual cycle. It also controls blastocystimplantation and uterine changes associatedwith early pregnancy (Figure 1). Disruption ofthe balance between angiogenic factors andtheir inhibitors at the time of implantation mayresult in first-trimester miscarriage or, alter-natively, defective placentation and therebyincreased risk of pregnancy-related disorders(Nardo, 2005).

ARTICLE IN PRESS

Figure 1. The expression of VEGF and VEGF-Rs during the folliculogenesis/oogenesis in menstrual (ovarian) cycle andfertilization processes. Expression levels of VEGF and VEGF-Rs are low during menstruation and the proliferation(estrogenic) phase and start to increase in the secretory phase. Expression of VEGF and its receptors reach highestlevels at the end of secretory (progestational) phase and ovulation. If no fertilization takes place, the expression ofVEGF and VEGF-Rs starts to decrease (A, and with darkness degree line of uterus). When fertilization occurs, VEGF andVEGF-Rs reach highest expression levels at implantation. Expression stays high during the post-implantation period(B, and with darkness degree line of uterus). PMF ¼ primordial follicles; PF1, 2 ¼ primary follicles (unilayered andmultilayered); SF ¼ secondary follicle (vesicular); GF ¼ Graafian follicle; COC ¼ cumulus oocyte complex; PO ¼ prim-ary oocyte; SO ¼ secondary oocyte; SP ¼ sperm; Th ¼ theca.

Vasculogenesis and angiogenesis during menstrual cycle and implantation 209

Hormonal control of endometrialangiogenesis

Endometrial growth and differentiation is underthe overall control of estrogen and progesterone.However, so far it is unclear how these steroidhormones specifically regulate endometrial angio-genesis. Tissue regression occurs following hormonewithdrawal at the end of the secretory phase,suggesting that angiogenesis required to repairthe vascular bed can occur in the absence ofsteroid hormones (Girling and Rogers, 2005).Detailed research is still required to determinenot only the mechanism(s) of angiogenesis atdifferent phases of the menstrual cycle, but alsohow each angiogenic event is regulated by steroidhormones.

Human chorionic gonadotrophin (hCG)

hCG has a direct role in regulating human implanta-tion and placentation: It stimulates VEGF expressionand thus indirectly causes increased angiogenesis(Licht et al., 2007). Exposure of human granulosa cellsto hCG stimulates the expression of VEGF mRNA(Neulen et al., 1995); and administration of hCG inwomen undergoing in vitro fertilization increasesurinary VEGF concentrations (Robertson et al., 1995)as well as serum and follicular fluid VEGF concentra-tions (Krasnow et al., 1996; Evans et al., 1998).

Estrogen

Most studies on endometrial angiogenesis havefocused on the effects of estrogen. There is current

ARTICLE IN PRESS

R. Demir et al.210

evidence that estrogen stimulates angiogenesis byacting directly on endothelial cells, and/or indir-ectly on other endometrial cell types via numerouspotential promoters.

Both progesterone and estrogen have been shownto induce VEGF expression in human uterinestromal cells (Hyder and Stancel, 1999; Zygmuntet al., 2003). Estradiol stimulates VEGF-A possiblyacting on two regions in the VEGF gene that arehomologous to untranslated and 30 estrogen-re-sponse elements. These regions are found in the 50

regions. Together, these elements confer respon-siveness to the estrogen receptors (ER-) a and b.

Both ER-a and b have been detected in endome-trial vascular smooth muscle cells (DiPietro andPolverini, 1993; Dunk et al., 2000), but only ER-bwas detected in endometrial endothelial cells, bothin vitro and in vivo. ER-a was only expressed at verylow levels, if at all (DiPietro and Polverini, 1993;O’Reilly et al., 1994). A splice variant of ER-b(isoform initially described as ER-b1, new isoformER-bcx/b2) was identified in endothelial cells acrossthe menstrual cycle in both functional and basallayers of the endometrium. The relative contributionof the different ER-b subtypes in regulating endome-trial angiogenesis remains to be elucidated. Con-ventionally, estrogen is accepted to be uterotrophicand to drive angiogenesis and vascular permeability.In in vitro assays, estradiol treatment stimulatedincreased human endometrial endothelial cell pro-liferation and the formation of angiogenic patterns(O’Reilly et al., 1994). Estradiol also increased theproliferative response of human endometrial en-dothelial cells to VEGF (Norrby et al., 1986). Duringthe estrogen-dominant proliferative phase of themenstrual cycle, elongation mechanisms of angiogen-esis have been demonstrated in the human endome-trium (Ferrara and Davis-Smyth, 1997).

Estrogen treatment of ovariectomized ewesresulted in an increased endometrial microvascularvolume density. Reynolds et al. (1992) hypothesizedthat vasodilatation accounts for a significantproportion of the increase in microvascular volumeseen in estrogen-treated sheep, but microvasculargrowth has also been described (Nehls and Drenc-khahn, 1995).

In contrast to the evidence suggesting thatestrogen can be angiogenic, recent data haveemerged suggesting that estrogen may also inhibitangiogenesis (Madjar et al., 1994). Ma et al.(2001) concluded that estrogen promotes vascularpermeability but profoundly inhibits endometrialangiogenesis. This conclusion was based partiallyon the reduction in the stromal area occupied byblood vessels two days after estrogen treatment ofovariectomized mice. Measuring changes in blood

vessel density, however, does not take into accountedema and resulting tissue expansion triggered byestrogen (Girling and Rogers, 2005). Heryanto et al.(2003) also observed a decrease in vascular densityin ovariectomized mice following estrogen treat-ment, as well as a reduction in the stromal celldensity illustrating that tissue edema had alsooccurred. However, the ratio of vascular densityto stromal cell density increased. This suggestedthat there was an increase in the number of vesselsrelative to the stromal compartment. In addition, asignificant increase in endothelial cell proliferationwas observed within 24 h of estrogen treatment(Kurjak et al., 1997; Francis et al., 1998).

Another study using ovariectomized mice did notshow any changes of the volume-fraction of total orproliferating endometrial endothelial cells 24 hafter estrogen treatment. These authors onlydescribed the formation of edema and an increasein uterine mass and endometrial area (Dennie et al.,1998). The discrepancy among these various studiesis difficult to explain, particularly as all used similarestrogen-treated ovariectomized mouse models. Itmay relate in part to the different approaches thatwere used to examine endometrial vasculature.

Progesterone

During the secretory phase of the human menstrualcycle, there is an increase in the vessel branch pointdensity, which may occur by sprouting or intussus-ceptive angiogenesis (Gambino et al., 2002).

Progesterone has been shown to inhibit endothe-lial cell proliferation in vitro; and to arrest the cellcycle in the G0/G1 phase in human dermalendothelial cells (Vazquez et al., 1999). Iruela-Arispe et al. (1999) identified progesterone recep-tors (isoforms not differentiated) in human endo-metrial endothelial cells and found thatprogesterone inhibited VEGF-induced endothelialcell proliferation.

In contrast, progesterone receptors (a or ß) werenot identified on endometrial endothelial cells invitro (Kayisli et al., 2004; Krikun et al., 2005;Kapiteijn et al., 2006). Despite a lack of receptors,progesterone treatment stimulated proliferation ofhuman endometrial endothelial cells in vitro andthe formation of angiogenic patterns (Kayisli et al.,2004). Although there are conflicting in vitroresults, endometrial angiogenesis has been ob-served in response to progesterone in vivo. In ratand mouse, considerable endothelial cell prolifera-tion was observed during early pregnancy, correlat-ing with increasing progesterone production by thecorpora lutea (Goodger and Rogers, 1993; Walter

ARTICLE IN PRESS

Vasculogenesis and angiogenesis during menstrual cycle and implantation 211

et al., 2005). In ovariectomized mice, progesteronetreatment resulted in an increased vascular density(Ma et al., 2001). Kayisli et al. (2004) suggesteddirect effects of estrogen and progesterone onhuman endometrial vascular endothelial cell func-tion. The latter cells show increased proliferativeand angiogenic activities in response to ovariansteroids (Kayisli et al., 2004).

Estrogen and progesterone have different effectsin vivo: Estrogen promotes uterine vascular perme-ability but profoundly stimulates angiogenesis,whereas progesterone also stimulates angiogenesiswith little effects on vascular permeability. Girlinget al. (2007) investigated the effects of estrogenand progesterone on endometrial vascular matura-tion in mice. They suggested that progesteronestimulates vessel maturation in the mouse endo-metrium, but not estrogen (Girling et al., 2007).These effects of estrogen and progesterone aremediated in concert with a differential spatiotem-poral expression of pro-angiogenic factors in theuterus (Ma et al., 2001).

Conclusions and prospects

Angiogenesis and vasculogenesis are initiated andmaintained by very complex signaling mechanisms.To understand the different molecular and functionalaspects of the two different processes, there is theneed for new experimental studies to better under-stand angiogenic mechanisms in the endometriumbased on angiogenic factors such as VEGF, EGF, PlGFand FGF. Different targeted gene knockout studies inmice and other animals will provide new data toelucidate the mechanisms by which these moleculescontribute to vasculogenic and angiogenic processesduring the menstrual cycle and implantation period.

Acknowledgments

This study was partly funded by TUBITAK and agrant from Akdeniz University Research Founda-tion. We would like to thank Leyla Sati and ZelihaS-ahin for their scientific collaboration.

References

Arroyo JA, Winn VD. Vasculogenesis and angiogenesis inthe IUGR placenta. Semin Perinatol 2008;32(3):172–7.

Augustin HG. Vascular morphogenesis in the ovary.Baillieres Best Pract Res Clin Obstet Gynaecol 2000;14(6):867–82.

Bagri A, Tessier-Lavigne M. Neuropilins as semaphorinreceptors: in vivo functions in neuronal cell migrationand axon guidance. Adv Exp Med Biol 2002;515:13–31.

Carmeliet P, Moons L, Luttun A, Vincenti V, CompernolleV, De Mol M, et al. Synergism between vascularendothelial growth factor and placental growthfactor contributes to angiogenesis and plasma extra-vasation in pathological conditions. Nat Med 2001;7(5):575–83.

Chung I, Yelian F, Zaher F, Gonik B, Evans M. Expressionand regulation of vascular endothelial growth factor ina first trimester trophoblast cell line. Placenta2000;21(4):320–4.

D’Alessandro S, Gelati M, Basilico N, Parati EA, HaynesRK, Taramelli D. Differential effects on angiogenesis oftwo antimalarial compounds, dihydroartemisinin andartemisone: implications for embryotoxicity. Toxicol-ogy 2007;241(1–2):66–74.

Demir R, Kayisli UA, Seval Y, Celik-Ozenci C, Korgun ET,Demir-Weusten AY, et al. Sequential expression ofVEGF and its receptors in human placental villi duringvery early pregnancy: differences between placentalvasculogenesis and angiogenesis. Placenta 2004;25(6):560–72.

Demir R, Kayisli UA, Cayli S, Huppertz B. Sequentialsteps during vasculogenesis and angiogenesis in thevery early human placenta. Placenta 2006;27(6–7):535–9.

Demir R, Seval Y, Huppertz B. Vasculogenesis andangiogenesis in the early human placenta. ActaHistochem 2007;109(4):257–65.

Dennie J, Mandeville JB, Boxerman JL, Packard SD, RosenBR, Weisskoff RM. NMR imaging of changes in vascularmorphology due to tumor angiogenesis. Magn ResonMed 1998;40(6):793–9.

Detillieux KA, Sheikh F, Kardami E, Cattini PA. Biologicalactivities of fibroblast growth factor-2 in the adultmyocardium. Cardiovasc Res 2003;57(1):8–19.

DiPietro LA, Polverini PJ. Angiogenic macrophages pro-duce the angiogenic inhibitor thrombospondin 1. Am JPathol 1993;143(3):678–84.

Djonov V, Baum O, Burri PH. Vascular remodeling byintussusceptive angiogenesis. Cell Tissue Res 2003;314(1):107–17.

Dunk C, Shams M, Nijjar S, Rhaman M, Qiu Y, Bussolati B,et al. Angiopoietin-1 and angiopoietin-2 activatetrophoblast Tie-2 to promote growth and migrationduring placental development. Am J Pathol 2000;156(6):2185–99.

Evans PW, Wheeler T, Anthony FW, Osmond C. Alongitudinal study of maternal serum vascular en-dothelial growth factor in early pregnancy. HumReprod 1998;13(4):1057–62.

Ferrara N. Vascular endothelial growth factor and theregulation of angiogenesis. Recent Prog Horm Res2000;55:15–35.

Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O’SheaKS, et al. Heterozygous embryonic lethality inducedby targeted inactivation of the VEGF gene. Nature1996;380(6573):439–42.

ARTICLE IN PRESS

R. Demir et al.212

Ferrara N, Davis-Smyth T. The biology of vascularendothelial growth factor. Endocr Rev 1997;18(1):4–25.

Folkman J, Klagsbrun M. Angiogenic factors. Science1987;235(4787):442–7.

Fong GH, Rossant J, Gertsenstein M, Breitman ML. Roleof the Flt-1 receptor tyrosine kinase in regulatingthe assembly of vascular endothelium. Nature 1995;376(6535):66–70.

Francis ST, Duncan KR, Moore RJ, Baker PN, Johnson IR,Gowland PA. Non-invasive mapping of placentalperfusion. Lancet 1998;351(9113):1397–9.

Gambino LS, Wreford NG, Bertram JF, Dockery P, Leder-man F, Rogers PA. Angiogenesis occurs by vesselelongation in proliferative phase human endome-trium. Hum Reprod 2002;17(5):1199–206.

Gargett CE, Rogers PA. Human endometrial angiogenesis.Reproduction 2001;121(2):181–6.

Girling JE, Rogers PA. Recent advances in endometrialangiogenesis research. Angiogenesis 2005;8(2):89–99.

Girling JE, Lederman FL, Walter LM, Rogers PA. Proges-terone, but not estrogen, stimulates vessel matura-tion in the mouse endometrium. Endocrinology2007;148(11):5433–41.

Goodger AM, Rogers PA. Uterine endothelial cell prolif-eration before and after embryo implantation in rats.J Reprod Fertil 1993;99(2):451–7.

Gordon JD, Shifren JL, Foulk RA, Taylor RN, Jaffe RB.Angiogenesis in the human female reproductive tract.Obstet Gynecol Surv 1995;50(9):688–97.

Heryanto B, Lipson KE, Rogers PA. Effect of angiogenesisinhibitors on oestrogen-mediated endometrial en-dothelial cell proliferation in the ovariectomizedmouse. Reproduction 2003;125(3):337–46.

Hickey M, Fraser IS. Clinical implications of disturbancesof uterine vascular morphology and function. Bail-lieres Best Pract Res Clin Obstet Gynaecol 2000;14(6):937–51.

Holash J, Wiegand SJ, Yancopoulos GD. New model oftumor angiogenesis: dynamic balance between vesselregression and growth mediated by angiopoietins andVEGF. Oncogene 1999;18(38):5356–62.

Huppertz B, Peeters LL. Vascular biology in implantationand placentation. Angiogenesis 2005;8(2):157–67.

Huppertz B, Abe E, Murthi P, Nagamatsu T, Szukiewicz D,Salafia C. Placental angiogenesis, maternal and fetalvessels – a workshop report. Placenta 2007;28(SupplA):S94–6.

Hyder SM, Stancel GM. Regulation of angiogenic growthfactors in the female reproductive tract by estrogensand progestins. Mol Endocrinol 1999;13(6):806–11.

Hyink DP, Abrahamson DR. Origin of the glomerularvasculature in the developing kidney. Semin Nephrol1995;15(4):300–14.

Iruela-Arispe ML, Rodriguez-Manzaneque JC, Abu-JawdehG. Endometrial endothelial cells express estrogen andprogesterone receptors and exhibit a tissue specificresponse to angiogenic growth factors. Microcircula-tion 1999;6(2):127–40.

Jakeman LB, Armanini M, Phillips HS, Ferrara N. Devel-opmental expression of binding sites and messenger

ribonucleic acid for vascular endothelial growth factorsuggests a role for this protein in vasculogenesis andangiogenesis. Endocrinology 1993;133(2):848–59.

Kaczmarek MM, Waclawik A, Blitek A, Kowalczyk AE,Schams D, Ziecik AJ. Expression of the vascularendothelial growth factor-receptor system in theporcine endometrium throughout the estrous cycleand early pregnancy. Mol Reprod Dev 2008;75(2):362–72.

Kapiteijn K, Koolwijk P, van der Weiden RM, van NieuwAmerongen G, Plaisier M, van Hinsbergh VW, et al.Human embryo-conditioned medium stimulates invitro endometrial angiogenesis. Fertil Steril 2006;85(Suppl 1):1232–9.

Kayisli UA, Demir R, Erguler G, Arici A. Vasodilator-stimulated phosphoprotein expression and its cyto-kine-mediated regulation in vasculogenesis duringhuman placental development. Mol Hum Reprod2002;8(11):1023–30.

Kayisli UA, Luk J, Guzeloglu-Kayisli O, Seval Y, Demir R,Arici A. Regulation of angiogenic activity of humanendometrial endothelial cells in culture by ovariansteroids. J Clin Endocrinol Metab 2004;89(11):5794–802.

Kayisli UA, Cayli S, Seval Y, Tertemiz F, Huppertz B, DemirR. Spatial and temporal distribution of Tie-1 and Tie-2during very early development of the human placenta.Placenta 2006;27(6–7):648–59.

Klagsbrun M, D’Amore PA. Regulators of angiogenesis.Annu Rev Physiol 1991;53:217–39.

Krasnow JS, Berga SL, Guzick DS, Zeleznik AJ, Yeo KT.Vascular permeability factor and vascular endothelialgrowth factor in ovarian hyperstimulation syndrome: apreliminary report. Fertil Steril 1996;65(3):552–5.

Krikun G, Schatz F, Taylor R, Critchley HO, Rogers PA,Huang J, et al. Endometrial endothelial cell steroidreceptor expression and steroid effects on geneexpression. J Clin Endocrinol Metab 2005;90(3):1812–8.

Kurjak A, Dudenhausen JW, Hafner T, Kupesic S, Latin V,Kos M. Intervillous circulation in all three trimesters ofnormal pregnancy assessed by color Doppler. J PerinatMed 1997;25(4):373–80.

Lambiase PD, Edwards RJ, Anthopoulos P, Rahman S,Meng YG, Bucknall CA, et al. Circulating humoralfactors and endothelial progenitor cells in patientswith differing coronary collateral support. Circulation2004;109(24):2986–92.

Licht P, Russu V, Lehmeyer S, Wissentheit T, SiebzehnrublE, Wildt L. Cycle dependency of intrauterine vascularendothelial growth factor levels is correlated withdecidualization and corpus luteum function. FertilSteril 2003;80(5):1228–33.

Licht P, Fluhr H, Neuwinger J, Wallwiener D, Wildt L. Ishuman chorionic gonadotropin directly involved in theregulation of human implantation? Mol Cell Endocrinol2007;269(1–2):85–92.

Ma W, Tan J, Matsumoto H, Robert B, Abrahamson DR, DasSK, et al. Adult tissue angiogenesis: evidence fornegative regulation by estrogen in the uterus. MolEndocrinol 2001;15(11):1983–92.

ARTICLE IN PRESS

Vasculogenesis and angiogenesis during menstrual cycle and implantation 213

Madjar H, Prompeler HJ, Sauerbrei W, Wolfarth R,Pfleiderer A. Color Doppler flow criteria of breastlesions. Ultrasound Med Biol 1994;20(9):849–58.

Maisonpierre PC, Suri C, Jones PF, Bartunkova S, WiegandSJ, Radziejewski C, et al. Angiopoietin-2, a naturalantagonist for Tie2 that disrupts in vivo angiogenesis.Science 1997;277(5322):55–60.

Malamitsi-Puchner A, Sarandakou A, Tziotis J, Stavreus-Evers A, Tzonou A, Landgren BM. Circulating angio-genic factors during periovulation and the luteal phaseof normal menstrual cycles. Fertil Steril 2004;81(5):1322–7.

Matsui N, Kawano Y, Nakamura S, Miyakawa I. Changes invascular endothelial growth factor production asso-ciated with decidualization by human endometrialstromal cells in vitro. Acta Obstet Gynecol Scand2004;83(2):138–43.

Meduri G, Bausero P, Perrot-Applanat M. Expression ofvascular endothelial growth factor receptors in thehuman endometrium: modulation during the menstru-al cycle. Biol Reprod 2000;62(2):439–47.

Nardo LG. Vascular endothelial growth factor expressionin the endometrium during the menstrual cycle,implantation window and early pregnancy. Curr OpinObstet Gynecol 2005;17(4):419–23.

Nehls V, Drenckhahn D. A novel, microcarrier-based invitro assay for rapid and reliable quantification ofthree-dimensional cell migration and angiogenesis.Microvasc Res 1995;50(3):311–22.

Neulen J, Yan Z, Raczek S, Weindel K, Keck C, Weich HA,et al. Human chorionic gonadotropin-dependent ex-pression of vascular endothelial growth factor/vascu-lar permeability factor in human granulosa cells:importance in ovarian hyperstimulation syndrome. JClin Endocrinol Metab 1995;80(6):1967–71.

Norrby K, Jakobsson A, Sorbo J. Mast-cell-mediatedangiogenesis: a novel experimental model using therat mesentery. Virchows Arch B Cell Pathol Incl MolPathol 1986;52(3):195–206.

O’Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA,Moses M, et al. Angiostatin: a novel angiogenesisinhibitor that mediates the suppression of meta-stases by a Lewis lung carcinoma. Cell 1994;79(2):315–28.

Otrock ZK, Makarem JA, Shamseddine AI. Vascularendothelial growth factor family of ligands andreceptors: review. Blood Cells Mol Dis 2007;38(3):258–68.

Patan S. Vasculogenesis and angiogenesis. Cancer TreatRes 2004;117:3–32.

Perrot-Applanat M, Ancelin M, Buteau-Lozano H, MeduriG, Bausero P. Ovarian steroids in endometrial angio-genesis. Steroids 2000;65(10–11):599–603.

Plaisier M, Rodrigues S, Willems F, Koolwijk P, vanHinsbergh VW, Helmerhorst FM. Different degrees ofvascularization and their relationship to the expres-sion of vascular endothelial growth factor, placentalgrowth factor, angiopoietins, and their receptors infirst-trimester decidual tissues. Fertil Steril 2007;88(1):176–87.

Poole TJ, Finkelstein EB, Cox CM. The role of FGF andVEGF in angioblast induction and migration duringvascular development. Dev Dyn 2000;220(1):1–17.

Pore N, Jiang Z, Gupta A, Cerniglia G, Kao GD, Maity A.EGFR tyrosine kinase inhibitors decrease VEGF expres-sion by both hypoxia-inducible factor (HIF)-1-indepen-dent and HIF-1-dependent mechanisms. Cancer Res2006;66:3197–204.

Postek A, Andronowska A, Chrusciel M, Jankowska K.Expression of VEGF-A, Flt-1, and Flk-1 in the arterialendothelial cells of the uterine broad ligamentthroughout the estrous cycle. Cell Tissue Res2007;330(2):313–9.

Rabbani ML, Rogers PA. Role of vascular endothelialgrowth factor in endometrial vascular events beforeimplantation in rats. Reproduction 2001;122(1):85–90.

Reynolds LP, Killilea SD, Redmer DA. Angiogenesis in thefemale reproductive system. FASEB J 1992;6(3):886–92.

Risau W. Mechanisms of angiogenesis. Nature 1997;386(6626):671–4.

Robertson D, Selleck K, Suikkari AM, Hurley V, Moohan J,Healy D. Urinary vascular endothelial growth factorconcentrations in women undergoing gonadotrophintreatment. Hum Reprod 1995;10(9):2478–82.

Seval Y, Korgun ET, Demir R. Hofbauer cells in earlyhuman placenta: possible implications in vasculogen-esis and angiogenesis. Placenta 2007;28(8–9):841–5.

Seval Y, Sati L, Celik-Ozenci C, Taskin O, Demir R. Thedistribution of angiopoietin-1, angiopoietin-2 andtheir receptors tie-1 and tie-2 in the very early humanplacenta. Placenta 2008;29(9):809–15.

Shalaby F, Rossant J, Yamaguchi TP, Gertsenstein M, WuXF, Breitman ML, et al. Failure of blood-islandformation and vasculogenesis in Flk-1-deficient mice.Nature 1995;376(6535):62–6.

Sherer D, Abulafia O. Angiogenesis during implantation,and placental and early embryonic development.Placenta 2001;22(1):1–13.

Shirayoshi Y, Yuasa Y, Suzuki T, Sugaya K, Kawase E,Ikemura T, et al. Proto-oncogene of int-3, a mouseNotch homologue, is expressed in endothelial cellsduring early embryogenesis. Genes Cells 1997;2(3):213–24.

Smith SK. Angiogenesis, vascular endothelial growthfactor and the endometrium. Hum Reprod Update1998;4(5):509–19.

Smith SK. Regulation of angiogenesis in the endome-trium. Trends Endocrinol Metab 2001;12(4):147–51.

Sugawara J, Mitsui-Saito M, Hayashi C, Hoshiai T, Senoo M,Chisaka H, et al. Decrease and senescence ofendothelial progenitor cells in patients with pree-clampsia. J Clin Endocrinol Metab 2005;90(9):5329–32.

Sugino N, Kashida S, Karube-Harada A, Takiguchi S, KatoH. Expression of vascular endothelial growth factor(VEGF) and its receptors in human endometriumthroughout the menstrual cycle and in early preg-nancy. Reproduction 2002;123(3):379–87.

Suri C, Jones PF, Patan S, Bartunkova S, Maisonpierre PC,Davis S, et al. Requisite role of angiopoietin-1, a

ARTICLE IN PRESS

R. Demir et al.214

ligand for the TIE2 receptor, during embryonicangiogenesis. Cell 1996;87(7):1171–80.

Tertemiz F, Kayisli UA, Arici A, Demir R. Apoptosiscontributes to vascular lumen formation and vascularbranching in human placental vasculogenesis. BiolReprod 2005;72(3):727–35.

Thurston G. Role of angiopoietins and Tie receptortyrosine kinases in angiogenesis and lymphangiogen-esis. Cell Tissue Res 2003;314(1):61–8.

Torry DS, Holt VJ, Keenan JA, Harris G, Caudle MR, Torry RJ.Vascular endothelial growth factor expression in cyclinghuman endometrium. Fertil Steril 1996;66(1):72–80.

Torry DS, Torry RJ. Angiogenesis and the expression ofvascular endothelial growth factor in endometriumand placenta. Am J Reprod Immunol 1997;37(1):21–9.

Ueno T, Nakamura T, Torimura T, Sata M. Angiogenic celltherapy for hepatic fibrosis. Med Mol Morphol 2006;39(1):16–21.

Valenzuela DM, Griffiths JA, Rojas J, Aldrich TH, Jones PF,Zhou H, et al. Angiopoietins 3 and 4: diverging genecounterparts in mice and humans. Proc Natl Acad SciUSA 1999;96(5):1904–9.

Vazquez F, Rodrıguez-Manzaneque JC, Lydon JP, EdwardsDP, O’Malley BW, Iruela-Arispe ML. Progesteroneregulates proliferation of endothelial cells. J BiolChem 1999;274(4):2185–92.

Warburton D, Schwarz M, Tefft D, Flores-Delgado G,Anderson KD, Cardoso WV. The molecular basis of lungmorphogenesis. Mech Dev 2000;92(1):55–81.

Walter LM, Rogers PA, Girling JE. The role of progester-one in endometrial angiogenesis in pregnant andovariectomised mice. Reproduction 2005;129(6):765–77.

Welter H, Wollenhaupt K, Tiemann U, Einspanier R.Regulation of the VEGF-system in the endometriumduring steroid-replacement and early pregnancy ofpigs. Exp Clin Endocrinol Diabetes 2003;111(1):33–40.

Weston G, Rogers PA. Endometrial angiogenesis. Bail-lieres Best Pract Res Clin Obstet Gynaecol 2000;14(6):919–36.

Zygmunt M, Herr F, Munstedt K, Lang U, Liang OD.Angiogenesis and vasculogenesis in pregnancy.Eur J Obstet Gynecol Reprod Biol 2003;110(Suppl 1):S10–8.