Implantation

Ji Yan

10-12-2012

• Infertility has been a serious worldwide problem, especially in developed countries. It has been reported that developed countries have much high infertility rate and much lower pregnancy rate (Cohen JE, 2010).

• The World Health Organization estimates that 8-12% of all couples world-wide experience infertility, at least 7.3 million people in the United States, 3.5 million people in the United Kingdom and 40 million couples in China suffer infertility.

• The World Health Organization concluded that in 47 developing countries (excluding China) 186 million women between the ages of 15-49 are infertile.

• Of the pregnancies that are lost, 75% represent a failure of implantation, and therefore are not clinically recognized as pregnancies.

• Implantation involves an intricate discourse between the embryo and uterus and is a gateway to further embryonic development. (NATURE REVIEWS | GENETICS, 2006 )

• Implantation is the process in which the blastocyst physically and physiologically comes into intimate contact with the uterus. (KevinYLee etal. 2004)

• Although implantation involves the interplay of numerous signaling molecules, the hierarchical instructions that coordinate the embryo-uterine dialogue are not well understood.

• A better understanding of peri-implantation biology could alleviate female infertility and help to develop novel contraceptives.

NATURE REVIEWS | GENETICS, 2006

• The successful implantation of an embryo is contigent upon cellular and molecular cross-talk between the uterus and embryo.

• Thus, deficiencies in uterine receptivity, embryo development, or the embryo-utrerine dialogue will compromise fertility.

Embryo

• The differentiated and expanded blastocyst is composed of three cell types: the outer polarized epithelial trophectoderm, the primitive endoderm, and the pluripotent ICM.

• The ICM provides the future cell lineages for the embryo

• The trophectoderm, the very first epithelial cell type in the developmental process, makes the initial physical and physiological connection with the uterine luminal epithelium.

• Trophoblast cells produce a variety of growth factors, cytokines, and hormones that influence the conceptus and maternal physiology in an autocrine, paracrine, and/or juxtacrine manner.

• Implantation is the process in which the blastocyst physically and physiologically comes into intimate contact with the uterus. (KevinYLee etal. 2004)

• The successful implantation of an embryo is contigent upon cellular and molecular cross-talk between the uterus and embryo.

• Thus, deficiencies in uterine receptivity, embryo development, or the embryo-utrerine dialogue wil compromise fertility.

Ovary

Oviduct

Cervix

Perimetrium

Myometrium

Endometrium

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• Fertilization occurs in the fallopian tube within 24 to 48 hours after ovulation.

• The initial stages of development, from fertilized ovum (zygote) to a mass of 12 to 16 cells (morula), occur as the embryo, passes through the fallopian tube.

• The morula enters the uterine cavity approximately two to three days after fertilization.

• The appearance of a fluid-filled inner cavity within the mass of cells marks the transition from morula to blastocyst and is accompanied by cellular differentiation.

The fertilization of an egg by a sperm, normally occurs in the fallopian tubes.

The next step for the fertilized egg is to implant into the walls of the uterus, beginning the initial stages of pregnancy.

If fertilization and/or implantation does not take place, the system is designed to menstruate (the monthly shedding of the uterine lining).

The events of implantation include: apposition of the blastocyst to the uterine lum

inal epitheliumadhesion to the epitheliumpenetration through the epithelium basal lami

na and invasion into the stromal vasculature

Figure 1. Blastocyst Apposition and Adhesion.The diagram shows a preimplantation-stage blastocyst (approximately six to seven days after conception) and the processesthought to be necessary for uterine receptivity and blastocyst apposition and adhesion.

Figure 2. Blastocyst Implantation.The diagram shows an invading blastocyst (about 9 to 10 days after conception) and the processes necessary for trophoblast invasion.

• Although various cellular aspects and molecular pathways of implantation process have been identified, a comprehensive understanding of the implantation process is still missing.

• A better understanding of the molecular signals that regulate uterine receptivity and implantation may lead to strategies to correct implantation failure and improve pregnancy rates.

• Recent advances in molecular and genetic approaches have led to the discovery of numerous molecules involved in embryo-uterine interactions; however, the precise sequence and details of the signaling cascades for many of these molecules have not yet been defined.

• We attempts to focus on a select number of signaling molecules and pathways that are implicated in embryo-uterine interactions in relation to implantation.

Steroid hormone signaling• Ovarian estrogen (E2) and progesterone

(P4) are essential for blastocyst implantation in the progesterone-primed uterus in mice and rats.

Estrogen action is normally considered to involve its interaction with a nuclear receptor (ER), a ligand-dependent transcription factor .

Progesterone receptor (PR)

adhesion molecules: cell-cell interactions

• selectins

• galectins

• Muc-1

• integrins

• cadherins

• trophinin

vasoactive factors

• It has long been speculated that vasoactive agents, such as histamine and PGs, are involved in many aspects of reproduction including ovulation, fertilization, implantation, and decidualization.

Histamine is a well-known neurotransmitter in the brain, but it is also involved in other physiological responses including gastric acid secretion, regulation of allergic reactions, and vascular permeability.

Because the process of implantation is considered a proinflammatory reaction and because increased vascular permeability at the site of blastocyst implantation is common to many species, it was suggested that histamine plays a role in implantation and decidualization .

• PGs possess vasoactive, mitogenic, and differentiating properties and are implicated in various female reproductive functions.

growth factors

• The expression of various growth factors and their receptors in the uterus in a temporal and cell-specific manner during the periimplantation period suggests that these factors are important for implantation.

growth factors

• EGF familyEGF, TGFα, HB-EGF, amphiregulin, betac

ellulin, epiregulin, neuregulin

• TGFβs

• FGFs

• IGFs

• PDGF

cytokines

TNFαIL-1M-CSFLIF

Homeobox genes • Hoxa-10 and Hoxa-11 are highly expresse

d in developing genitourinary tracts and the adult female reproductive tract, suggesting roles in reproductive events.

• Hmx3 is primarily expressed in the myometrium during early pregnancy.

• In brief, implantation in mammals absolutely depends upon synchronized development of the blastocyst to the stage when it is competent to implant and the uterus to the stage when it is receptive to blastocyst growth and implantation.

• Ovarian estrogen (E2) and progesterone (P4) are the primary effectors that direct the prereceptive uterus to a receptive state via a number of locally expressed growth factors, cytokines, transcription factors,and vasoactive mediators in the uterus.

FIG. 1. A scheme of signaling networks in embryo-uterine communication during implantation in mice.

Le, Luminal epithelium; Ge, glandular epithelium;S, stroma;CE, catechol estrogen.

• Tissue remodeling and angiogenesis are two hallmark events during implantation and decidualization.

• Increased vascular permeability and angiogenesis are crucial to successful implantation, decidualization, and placentation.

Vascular epithelial growth factor (VEGF)

• VEGF, originally discovered as a vascular permeability factor, is also a potent mitogen for endothelial cells and a key regulatory growth factor for vasculogenesis and angiogenesis.

• Targeted disruption of even one allele of the Vegf gene results in embryonic death in uteri during midgestation with aberrant blood vessel formation.

Angiogenic signaling in the uterus during implantation

• The proangiogenic factor VEGF and its receptor Flt1 (VEGFR1) and Flk1 (VEGFR2) are primarily important for uterine vascular permeability and angiogenesis before and during the attachment phase of the implantation process.

• Among the factors regulating uterus development, vascular remodeling promoters are critical for uterus function and fertility. VEGF as a major factor promotes endothelial cell growth and blood vessel development.

• In addition, VEGF has been found playing major roles in non-endothelial cells.

• Gene expression studies and genetically engineered mouse models have provided valuable clues to the implantation process with respect to specific growth factors, cytokines, lipid mediators, adhesion molecules, and transcription factors.

• A staggering amount of information from microarray experiments is also being generated at a rapid pace. If properly annotated and explored, this information will expand our knowledge regarding yet-to-be-identified unique, complementary, and/or redundant molecular pathways in implantation.

(Endocrine Reviews 25: 341–373, 2004)

• High-throughput sequencing technology has been used for transcriptone analysis .

• Taking advantage of the Illumina Genome Analyzer platform, we performed transcriptional profiling on the VEGF-repressed mouse uterus.

• The Digital Gene Expression (DGE) tag profiling allows us to quantify gene expression levels of relative samples.

• In this study, a genetically repressed VEGF

mouse model is used to profile uterus transcriptome at post coitus 2.5.

• Go analysis of uterus expressed genes, uterus specific genes and VEGF-reguated genes, and their related signaling pathways were analyzed.

• The Gene Ontology project provides an ontology of defined terms representing gene product properties. The ontology covers three domains:– cellular component, the parts of a cell or its extracellular

environment; – molecular function, the elemental activities of a gene

product at the molecular level, such as binding or catalysis;

– biological process, operations or sets of molecular events with a defined beginning and end, pertinent to the functioning of integrated living units: cells, tissues, organs, and organisms.

Gene ontology (GO)

KEGG pathway

• KEGG (Kyoto Encyclopedia of Genes and Genomes) is a collection of databases dealing with genomes, enzymatic pathways, and biological chemicals.

• The PATHWAY database records networks of molecular interactions in the cells, and variants of them specific to particular organisms.

Up-regulated genes

Down-regulated genes

A: Up-regulated genesB: Down-regulated genes

• To determine whether VEGF regulate uterus development through antisense genes expression, the antisense transcript levels were analyzed.

• Antisense expression levels showed correlation with sense expression which may indicate non-specific transcription near promoters and enhancers.

Genes relatived to muscle devolpment

• Enzymes: Sult1d1, Ctla2a, Hdc, Pkdcc, Pcx, Gamt, Usp2, Mylk, Car2, Mif, Rnf187

• Receptors: P2ry14, Usmg5, Ramp3, Oxtr, Mt2, F2r, Sfrp2, Ly6e,Fgfl1

• Structure proteins: Acta2, Actg2, Eln, Gjb2, Utrn, Myh11

• Glycoprotein: Inhbb, CD24a, Tnc, Ihh• Transcript factors: Hopx, Tcf23, Lef1, Elf3,

Skil• Regulatory proteins: Cnn1, Cabl1, Fxyd4

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