ArticlePDF AvailableLiterature Review

Wang H, Dey SK. Roadmap to embryo implantation: clues from mouse models. Nat Rev Genet 7: 185-199

April 2006
Nature Reviews Genetics 7(3):185-99

April 2006
7(3):185-99

DOI:10.1038/nrg1808

Source
PubMed

Authors:

Haibin Wang

School of Medicine, Xiamen University, Xiamen China

Sudhansu K Dey

Cincinnati Children's Hospital Medical Center

Implantation involves an intricate discourse between the embryo and uterus and is a gateway to further embryonic development. Synchronizing embryonic development until the blastocyst stage with the uterine differentiation that takes place to produce the receptive state is crucial to successful implantation, and therefore to pregnancy outcome. Although implantation involves the interplay of numerous signalling molecules, the hierarchical instructions that coordinate the embryo-uterine dialogue are not well understood. This review highlights our knowledge about the molecular development of preimplantation and implantation and the future challenges of the field. A better understanding of periimplantation biology could alleviate female infertility and help to develop novel contraceptives.

Content uploaded by Haibin Wang

Content may be subject to copyright.

Departments of Pediatrics,

Cell & Developmental

Biology, and Pharmacology,

Division of Reproductive and

Developmental Biology,

Vanderbilt University Medical

Center, Nashville, Tennessee

37232, USA.

Correspondence to S.K.D.

e-mail: sk.dey@vanderbilt.edu

doi:10.1038/nrg1808

Blastocyst

An embryonic stage in

mammals that is derived from

a morula and is comprised of a

fluid-filled cavity (blastocoel)

and two cell types, the inner cell

mass and the trophectoderm.

Roadmap to embryo implantation:

clues from mouse models

Haibin Wang and Sudhansu K. Dey

Abstract | Implantation involves an intricate discourse between the embryo and uterus and

is a gateway to further embryonic development. Synchronizing embryonic development

until the blastocyst stage with the uterine differentiation that takes place to produce the

receptive state is crucial to successful implantation, and therefore to pregnancy outcome.

Although implantation involves the interplay of numerous signalling molecules, the

hierarchical instructions that coordinate the embryo–uterine dialogue are not well

understood. This review highlights our knowledge about the molecular development of

preimplantation and implantation and the future challenges of the field. A better

understanding of periimplantation biology could alleviate female infertility and help to

develop novel contraceptives.

The implantation of the blastocyst into the maternal

uterus is a crucial step in mammalian reproduction

and, like many developmental processes, it involves

an intricate succession of genetic and cellular interac-

tions, all of which must be executed within an optimal

time frame. In mammals, the beginning of new life is

seeded at fertilization. The fertilized egg undergoes

many cell divisions to form a blastocyst (

BOX 1, part a).

These developmental events are synchronized with the

proliferation and differentiation of specific uterine cell

types, primarily under the direction of ovarian oestro-

gen and progesterone (P

). These hormones make the

uterus conducive (‘receptive’) to accept a blastocyst for

implantation

1–4

(BOX 2).

A reciprocal interaction between the blastocyst and

receptive uterus is essential for implantation. Early preg-

nancy loss in humans, which often occurs due to defects

that occur before, during or immediately after implan-

tation, is a worldwide social and economic concern.

Although the human population is growing rapidly and

will probably reach nine billion by 2050, 15% of couples

worldwide are childless because of infertility. Many under-

lying causes of human infertility have been overcome by

in vitro fertilization and embryo-transfer techniques;

implantation rates, however, remain disappointingly

low, probably owing to embryos being transferred into

a nonreceptive uterus. There is, therefore, a continuing

need to unravel the complexities of preimplantation

embryonic development and implantation to address

two contrasting global issues: to improve infertility,

and to develop novel contraceptives.

Here, we focus on the molecular and genetic mecha-

nisms of implantation that have been gleaned primarily

from mouse models, and which could be relevant to

humans. We describe the signalling networks that direct

preimplantation embryonic development, confer blas-

tocyst competency and uterine receptivity to implanta-

tion, instigate the blastocyst–uterine dialogue at various

phases of implantation, and finally, participate in orient-

ing the embryo–uterine axis during the postimplanta-

tion period. The clinical implications of these findings

are also illustrated.

Revisiting this field is timely because of the emergence

of technological advances that allow us, for example, to

profile global gene and protein expression in the embryo

and uterus, and to predict how molecules interact during

implantation. Many gene-knockout mouse models have

also provided a wealth of information that needs to be

carefully interpreted in addressing human fertility.

Although the cellular events that define the various

stages of implantation have been described

1,5

, the molecu-

lar genetic pathways that are crucial to this process, and

how they interact, are not clearly understood. Because

this complex process varies across species (

BOX 1, part b)

the formulation of a unified model for the molecular basis

of implantation in mammals seems unrealistic at present.

However, inroads can be made by addressing a few crucial

questions to determine: which signalling pathways are

crucial; which are complementary or antagonistic; and

how these pathways are coordinated. Another challenge

is to identify the signalling pathways that have a limited

role during normal pregnancy, but that become important

REVIEWS

NATURE REVIEWS

GENETICS VOLUME 7

MARCH 2006

185

Oviduct

E1.0

E1.5

E2.0

E2.5

E3.0

2 cells

4 cells

8 to 16-cell

compacted

morula

Early

blastocyst

Late

blastocyst

Implantation

Uterus

Oocyte

Ovulation

Fertilized

egg

(zygote)

Fertilization

E0.5

Zona pellucida

ICM

E4.0

Trophectoderm

Epiblast

Primitive endoderm

Blastocoel

Inner cell mass

Ovary

8-cell

uncompacted

embryo

RabbitGuinea pig PrimateMouse and rat

under conditions of stress. Answers to these questions

might help to improve fertility and fertility-associated

issues in

women.

Preimplantation embryonic development

The development of the mammalian preimplantation

embryo encompasses the period from fertilization to

implantation. This period is marked by three princi-

pal transitions, all of which involve dynamic genetic

programming: fertilization and the first cell division;

continued cell division; the establishment of cell polarity

and

compaction to form a morula; and lineage differentiation

to form a blastocyst (followed by implantation).

At the blastocyst stage, embryos mature and escape

from their outer shells

(zona pellucidae) and then gain

implantation competency. The mature blastocyst

is composed of three cell types: the outer epithelial

trophectoderm (Tr), the primitive endoderm (PE) and

the pluripotent

inner cell mass (ICM) (BOX 1, part a). The

ICM generates future cell lineages of the embryo proper,

Box 1 | Preimplantation and implantation events

| Preimplantation embryo development and implantation in mice. Following fertilization in the oviduct, the embryo

undergoes several rounds of mitotic cell division, ultimately forming a ball of cells called a morula. At the late morula

stage, the embryo enters the uterine lumen and transforms into a blastocyst that contains a cavity (called blastocoel) with

two distinct cell populations, the inner cell mass (ICM) and the trophectoderm (the progenitor of trophoblast cells).

Before implantation, the blastocyst escapes from its outer shell (the zona pellucida) and differentiates to produce

additional cell types — the epiblast and the primitive endoderm. At this stage, the trophectoderm attaches to the uterine

lining to initiate the process of implantation. E, embryonic day.

b | Implantation strategies in different species. The main

purpose of implantation is to ensure that trophoblast cells firmly anchor into the endometrial stroma. Ultrastructural

studies have revealed that there are different modes of implantation in mammals : the trophectoderm-derived

trophoblast (T) cells can breach the uterine luminal epithelium, coalesce with it or trespass between the uterine cells to

home in on the underlying stroma. In mice and rats, the attachment of the blastocyst (represented by En, the embryonic

endoderm) to the luminal epithelium (LE) imparts epithelial apoptosis locally at the site of attachment, facilitating the

penetration of trophoblast cells through the LE layer into the stroma (S). In guinea pigs, the

syncytial trophoblast makes

focal protrusions through the zona pellucida (ZP) and intrudes between epithelial cells, ultimately embedding the

embryo in the uterine stroma.

In rabbits, clusters of trophoblast cells (trophoblastic knobs) fuse with the LE (T–LE fusion)

to form symplasma. In primates, the syncytial trophoblast is formed near the ICM, which intrudes between uterine

epithelial cells and penetrates the basal lamina. D, decidual cells. Part

a is adapted with permission from REF. 88 © (2001)

Terese Winslow. Part

b is adapted with permission from REF. 1 © (2000) Elsevier Science.

Compaction

An embryonic state in which

the cells of the morula are

flattened and cell outlines

are not clearly distinguishable.

Morula

A cluster of blastomeres that

results from the early cleavages

of a zygote.

Zona pellucida

An outer shell composed of

glycoproteins that encircles

oocytes or preimplantation

embryos.

Trophectoderm

The outer layer of the

blastocyst that is the progenitor

of future trophoblast cell types.

Inner cell mass

Cells that are present inside

the blastocyst. These cells are

pluripotent and give rise to the

embryo proper (that is, the

cells that are not destined to

become the placenta).

Syncytial trophoblast

The syncytial multinucleated

outer layer of the trophoblast.

REVIEWS

186

MARCH 2006

VOLUME 7 www.nature.com/reviews/genetics

Proestrus

–14 –7 0 7 10 14

Pre-receptive

Proliferative phase Secretory phase

Receptive Refractory

Mouse

Human

Day of pregnancy or pseudopregnancy

Day of cycle

Receptive Refractory

123456

mRNA differential display

A technique for detecting

genes that are expressed only

under specific conditions; it

involves isolating mRNA from

two or more cell populations

and comparing their transcript-

expression levels.

Pseudopregnancy

A condition similar to

pregnancy, without the

presence of a fertilized egg,

which is produced by sterile

mating or hormone treatment.

while the Tr makes the first physical and physiological

connection with the luminal epithelium (LE) of the

maternal uterus for implantation.

Fertilization and first cell division: maternal to zygotic

expression. A unique feature of preimplantation embry-

onic development is the presence of maternally stored

RNAs and proteins in mature, unfertilized eggs. In mice,

fertilization triggers the degradation of oocyte-stored

transcripts, which is 90% complete by the 2-cell stage

Transcription from the newly formed zygotic genome,

known as zygotic genome activation (ZGA), establishes

the gene-expression patterns that are required for con-

tinued development. A comprehensive molecular charac-

terization of the developmental reprogramming from

maternal to zygotic gene expression has been hindered by

the scarcity of embryonic tissues and lack of appropriate

molecular approaches. Conventional methods, such as

reverse-trancriptase PCR, western blotting and immuno-

histochemistry have been used to examine the expression

patterns of a limited number of genes, but not the dynamic

changes that occur during early development. To assess

more robust gene-expression patterns, high-resolution 2D

protein gels

7, 8

, mRNA differential display

and the analysis

of expressed sequence tags (ESTs) that were derived from

libraries of several preimplantation stages

were carried

out. However, these studies provided information on

signature transcripts and/or proteins, but not global gene

expression or proteome profiles.

Recently, global gene-expression profiles that were

derived from microarray experiments have gener-

ated a comprehensive data set that covers nearly

all mouse genes during preimplantation develop-

ment

11–13

. One remarkable finding is the existence

of programmed waves of upregulated and downregu-

lated gene expression, which parallels the stages of

embryonic development. According to Hamatani et al.

maternal-to-zygotic gene activation shows two

principal transient waves of de novo transcription.

The first wave corresponds to the ‘major ZGA’, which

peaks between the 2- and 4-cell stages and leads to

the most marked genetic reprogramming. The second

wave, mid-preimplantation gene activation (MGA),

peaks at the 8-cell stage and precedes the morula-to-

blastocyst formation

(FIG. 1a)

; indeed, MGA involves

the expression of intercellular adhesion molecules dur-

ing blastomere polarity and compaction. Irrespective of

the underlying mechanisms, the identification of zygotic

transcription cascades that occur at each successive

phase (minor ZGA>major ZGA>MGA;

FIG. 1a) is the

first step towards analysing the complex gene regulatory

network that governs embryonic development.

Studies of transgenic mice show that many genes have

vital functions in preimplantation embryonic devel-

opment, and that their functions, as inferred by gene

targeting, are consistent with their gene-expression pro-

files (see

Supplementary information S1 (table); FIG. 1a).

For example, the maternal-effect gene Mater is detected

Box 2 | The window of uterine receptivity in mice and humans

In placental mammals, the uterus differentiates into an altered state when

implantation-competent blastocysts are ready to initiate implantation. This

state is called uterine receptivity for implantation and lasts for a limited

time. At this stage, the uterine environment is conducive to blastocyst

growth, attachment and the subsequent events of implantation. The major

hormones that specify uterine receptivity are the ovarian steroids

progesterone (P

) and oestrogen (E

). In mice, the oestrous cycle is short

(~4 days) and often irregular. Therefore, it is difficult to determine the

receptive phase during the cycle. Blastocyst transfers in pseudopregnant

recipients were used to determine various phases of uterine sensitivity to

implantation. In contrast, the menstrual cycle in women is long and the

hormonal changes are more predictable, which allows the state of uterine

receptivity to be determined. A surge of leutinizing hormone (LH), which is

secreted from the pituitary, is essential to ovulation and in programming

the secretion of oestrogen and progesterone by the ovary.

Uterine sensitivity to implantation is classified into prereceptive,

receptive and nonreceptive (refractory) phases. During the prereceptive

phase, the uterus is unable to initiate implantation, but the uterine

environment is less hostile to blastocyst survival. In contrast, during the

refractory phase, the uterine environment is unfavourable to blastocyst

survival. In mice (top diagram), the uterus is receptive on day 4 of

pregnancy or

pseudopregnancy, whereas it is prereceptive on days 1–3,

and by the afternoon of day 5 it becomes nonreceptive (refractory) to

implantation.

In humans (bottom diagram), the uterus is classified histologically and

functionally into proliferative (follicular) and secretory (luteal) phases

during the average 28–30-day menstrual cycle. During the secretory phase,

the uterus is considered prereceptive for the first ~7 days following

ovulation (day 0). The uterus then becomes receptive during the mid-

secretory phase, which spans 7–10 days after ovulation; the nonreceptive

(refractory) phase comprises the rest of the secretory phase.

REVIEWS

NATURE REVIEWS

GENETICS VOLUME 7

MARCH 2006

187

Unfertilised 1-cell 2-cell 4-cell 8-cell Morula Blastocyst

Degradation of

maternal mRNAs

Embryonic–abembryonic axis

Inner cell mass

(pluripotent)

Precursor

(totipotent)

Nanog

Gata6

Oct4

Cdx2

Trophectoderm

(multipotent)

ICM

Primitive endoderm

(multipotent)

Epiblast

(pluripotent)

Minor

ZGA

Major

ZGA

MGA

Morula

compaction

Blastocyst

cavitation

Embryonic region

including ICM cells

Extraembryonic cells

interacting with uteri

during implantation

Boundary

Blastocyst Late blastocystLate morula

Cdx2

Oct4/Nanog

Oct4

Gata6

only in mature, unfertilized eggs, and its deletion

restricts development beyond the 2-cell stage

. Other

studies have identified additional preimplantation

maternal and zygotic genes (see

Supplementary infor-

mation S1

(table); FIG. 1a). Collectively, these studies

have advanced our knowledge of preimplantation

embryonic development, but a comprehensive under-

standing of embryonic development, especially in

humans, is far from complete.

Cell polarity.

Preimplantation development involves the

transition of totipotent, fertilized eggs to blastocysts that

contain both pluripotent ICM cells and the Tr, which

is the progenitor of the trophoblast. One fundamental

question, discussed in this section, is how the cellular

polarity of the embryonic–abembryonic (Em–Ab) axis

is established with the formation of a blastocyst

(FIG. 1b).

The traditional opinion is that embryonic develop-

ment is symmetrical before implantation because each

blastomere in 8- or 16-cell mouse embryos can produce

an offspring. However, recent studies have revealed

asymmetries in the potential of cells, even at very early

stages

(BOX 3).

Lineage differentiation. Irrespective of the debate about

polarity, the molecular crosstalk that segregates and

differentiates the ICM and Tr lineages in blastocysts

is essential to implantation because it is the Tr that

initiates this process together with the LE. Microarray

and deletion studies have identified several genes that

are crucial for these two cell-lineage segregations (see

Supplementary information S1 (table)), which include

those that encode many transcription factors, such as

OCT4, SOX2, NANOG, CDX2

and Eomesodermin

(EOMES)

15, 16

(FIG. 1b,c).

ICM formation depends on OCT4, which is encoded

by Pou5f1, as Pou5f1-mutant blastocysts contain only Tr.

Pou5f1 is restricted to the ICM at the blastocyst

stage

. SOX2, a high-mobility group (HMG)-box

transcription factor, shows a similar expression pro-

file to Pou5f1, and together they prevent trophoblast

specification

. However, the inability of OCT4 alone

to maintain

embryonic-stem-cell (ES cell) pluripotency

in the absence of leukaemia inhibitory factor (LIF)

indicates that other pluripotency-promoting factors,

such as NANOG (a homeobox (Hox) transcription

factor), are involved. The consensus is that, while both

NANOG and OCT4 are required for ICM specifica-

tion, they suppress the formation of extraembryonic

lineages; OCT4 represses trophoblast and NANOG

parietal–visceral endoderm formation

20,21

. A recent

genome-scale location analysis in human ES cells has

revealed a novel mechanism for establishing pluripo-

tency. It showed that OCT4, SOX2 and NANOG co-

occupy a substantial portion of their target genes and

collaborate to form a circuitry of autoregulatory and

feed-forward loops

It has been proposed that the Tr develops by default

in the absence of OCT4. However, CDX2, a caudal-type

homeodomain protein, is crucial for segregating ICM

and Tr lineages at the blastocyst stage by ensuring the

Figure 1 | Genes governing the development of the preimplantation mouse

embryo. a | Gene expression during preimplantation embryo development. The

diagram depicts the waves of gene expression that occur in preimplantation embryos,

based on microarray studies, from the degradation of maternal genes, to the three

successive waves of overlapping zygotic gene expression (minor and major ZGA

(zygotic genome activation), and MGA (mid-preimplantation gene activation)) —

following the MGA, genes that are specific to morula compaction and blastocyst

cavitation are also expressed in a wave-like fashion. In addition, many genes that are

expressed in embryos and stem cells are crucial to preimplantation development

(see

Supplementary information S1 (table)). b,c | Genes that are crucial for cell-fate

and lineage segregations during the morula-to-blastocyst transition, as determined by

expression and mutation studies in mice. Panel (b) represents the gene-expression

patterns during blastocyst formation. Whereas Oct4 (dark pink) is expressed throughout

the embryo before the late morula stage, the expression of Nanog (light pink) is

specifically induced in the inside cells of late morulae. Cdx2 (blue) is expressed in the

outer layer of cells in late morulae and is required for the repression of Oct4 and Nanog

in the trophectoderm (Tr) of the blastocyst. Oct4 is crucial for inner-cell-mass (ICM)

formation. Gata6 (green) is expressed in the primitive endoderm of the late blastocyst,

where Oct4 and Nanog are repressed. Panel (c) shows the genetic model of lineage

decision. Oct4 represses Cdx2 expression, which in turn represses Oct4 expression to

allow segregation of the ICM and Tr lineages of the blastocyst. An antagonism between

Nanog and Gata6 segregates epiblast and primitive endoderm within the ICM. Panel

(a) is adapted with permission from

REF. 11 © (2004) Elsevier Science. Panel (b) is

adapted with permission from

REF. 113 © (2005) Blackwell Publishing Ltd.

REVIEWS

188

MARCH 2006

VOLUME 7 www.nature.com/reviews/genetics

Cavitation

The creation of a hollow space

that appears within the early-

cleaving embryos to form a

blastocyst.

Embryonic stem cells

(ES cells). Stem cells have the

dual capacity to self-replicate

and differentiate into several

specialized derivatives. ES cells

are pluripotent cells that are

derived from pre-implantation-

stage (usually blastocyst)

mammalian embryos. Mouse

ES cells can be propagated

and manipulated in vitro, yet

still retain their pluripotency.

Polar body

The structure that is extruded

from the oocyte during

meiosis, which contains one

haploid set of chromosomes.

Window of implantation

A limited time period when

the uterine environment is

conducive to supporting

blastocyst growth, attachment

and the subsequent events of

implantation.

Delayed implantation

A state of suspended animation

of the blastocyst, characterized

by halted growth and

postponement of implantation.

In mice, ovariectomy on day 4

morning of pregnancy, before

ovarian oestrogen secretion,

initiates blastocyst dormancy,

which can last for many days if

treated with P

; an oestrogen

injection rapidly activates

blastocysts and initiates their

implantation.

Blastocyst activation

The event that leads to the

competency of the blastocyst

to implant.

repression of OCT4 and NANOG in the Tr (FIG. 1b,c).

This was shown by the finding that CDX2 deficiency

results in a failure to downregulate

Oct4 and Nanog in

the outer cells of the blastocyst, which results in the

loss of the epithelial integrity of these cells and their

ultimate demise

. Another gene that is involved in Tr

development is that encoding the T-box transcription

factor EOMES, which, like

Cdx2, is expressed in the Tr

However, embryos that lack

Eomes develop to blastocysts

and correctly express Cdx2 and Oct4 in Tr and ICM cells,

respectively

. It is suggested that Cdx2 is the earliest

inducer of the Tr lineage in late morulae, whereas Eomes

is required for Tr proliferation and differentiation at the

blastocyst stage.

Determinants of blastocyst competency

For successful implantation to occur in the receptive

uterus, the blastocyst must also attain implantation

competency. The first evidence that the state of activity

of the blastocyst determines the

‘window’ of implantation

in the receptive uterus was derived from reciprocal

blastocyst-transfer experiments in a

delayed-implantation

mouse model

25,26

. This model is a powerful tool for

defining the molecular signalling components that direct

blastocyst activation or dormancy. Nearly 100 mammals in

seven orders undergo delayed implantation

27,28

, but the

underlying mechanism remains largely unknown. Using

this model, a global gene-expression study showed that

these two different physiological states of the blastocyst

are molecularly distinguishable

. The main functional

categories of altered genes include cell-cycle, cell-

signalling and energy-metabolic pathways. This study

also showed an upregulated expression of Hegf1 (which

encodes heparin-binding EGF-like growth factor (

HB-

EGF

)) in activated blastocysts, a finding that is comple-

mentary to earlier reports of upregulated expression of

its receptors

ErbB1 and ErbB4 in similar blastocysts

29–31

Other signalling molecules also participate in blas-

tocyst dormancy and activation. There is evidence that

catecholoestrogens that are produced from primary

oestrogens in the uterus activate blastocysts

. Another

lipid-signalling molecule that targets blastocysts is

an endocannabinoid anandamide, which activates

G-protein-coupled cannabinoid receptors CB1 and CB2.

Expression of

Cb1 in the Tr, and uterine synthesis of anan-

damide, indicate that endocannabinoid signalling is crucial

to implantation in mice

33–35

. Levels of uterine anandamide

and blastocyst CB1 are coordinately downregulated with

the attainment of uterine receptivity and blastocyst acti-

vation, respectively, in contrast to their elevated levels in

the nonreceptive uterus and dormant blastocysts

33,36,37

Indeed, implantation is postponed in wild-type mice that

are maintained on sustained levels of exogenously admin-

istered cannabinoid ligands, an effect that depends on the

expression of CB1 receptors on the embryo

. Anandamide

regulates blastocyst function by differentially modulating

mitogen-activated protein kinase (MAPK) signalling and

-channel activity via CB1 (REF. 36). This is consist-

ent with findings that MAPK and phosphatidylinositol

3-kinase/Ca

-signalling cascades are crucial to blastocyst

development and activation

38–41

Most gene-expression studies have so far pointed

towards changes in Tr gene expression during blastocyst

dormancy or activation. It remains to be seen whether

gene expression in the ICM also changes with the state

of activity of the blastocyst. A greater insight into the

molecular basis of blastocyst competency for implanta-

tion might help to improve pregnancy rates in human

IVF programs.

Determinants of uterine receptivity

Molecular and genetic evidence indicates that locally

produced signalling molecules, including cytokines,

growth factors, homeobox transcription factors, lipid

mediators and morphogens, together with ovarian

hormones, serve as autocrine, paracrine and juxta-

crine factors to specify uterine receptivity

(TABLES 1,2).

In this section, evidence is presented for a novel signal-

ling network that involves cytokines, homeotic proteins

and morphogens in implantation.

Box 3 | Cell polarity in the early embryo: an ongoing debate

Specific labelling of blastomeres or zona pellucidae indicates that the plane of the first cleavage specifies embryonic

polarity. That is, the plane of cleavage is orthogonal to the future embryonic–abembryonic axis of the blastocyst, with one

blastomere predominantly contributing to the embryonic pole (polar trophectoderm (Tr) and deeper inner-cell-mass (ICM)

cells) and the other to the abembryonic pole (mural Tr and more superficial ICM)

89–93

. This indicates a developmental

asymmetry at the 2-cell stage, which is supported by lineage-tracing experiments

94–96

, and by observations that 2-cell

embryos divide asynchronously, with daughter cells making a differential contribution to the future ICM or Tr

97,98

. However,

the observation that both blastomeres in 2-cell embryos make a similar contribution to all cell types during the

postimplantation development of embryos that harbour a Cre-reporter indicates that there is no absolute programming of

lineage segregation for either the ICM or Tr at the 2-cell stage

. This is consistent with studies that used fluorescent-tracer

labelling and time-lapse imaging

99–101

. Collective analysis provides evidence that polarity with lineage differentiation is

first clearly discernible with the onset of blastocyst formation

101

Another question in this ongoing debate is whether the first cleavage occurs randomly or is prepatterned

102,103

. It was

proposed that the sperm-entry site, along with the second

polar body from the meiotic division, marks the first cleavage

plane

92,104,105

. However, the results of the labelling of internalized sperm components

106

, and the fact that the polar body is

not stationary but can move to the cleft between two blastomeres after cleavage

107

, challenge this view. Time-lapse

recordings show that the first cleavage plane is not predetermined, but is defined by the apposition of two pronuclei at the

centre of the zygote

107

. The use of videomicroscopy to visualize the mitotic spindle using GFP-labelled tubulin also shows

that the cleavage plane is randomly oriented in 2-cell embryos, arguing against prepatterning before embryonic

compaction

108

. Therefore, the question of the origin of cell polarity and lineage differentiation is still under debate.

REVIEWS

NATURE REVIEWS

GENETICS VOLUME 7

MARCH 2006

189

Oestrogen and progesterone. The principal hormones

that direct uterine receptivity are ovarian P

and oes-

trogen

. P

is essential for implantation and pregnancy

maintenance in all mammals studied, whereas the

requirement for ovarian oestrogen is species-specific

For example, ovarian P

and oestrogen are essential to

implantation in mice and rats, but ovarian oestrogen is

dispensable in pigs, guinea pigs, rabbits and hamsters.

However, oestrogen that is produced by embryos is

considered important for implantation in these last

four species

; whether ovarian or embryonic oestrogen

participates in human implantation remains unknown.

The uterine effects of oestrogen and P

are primarily

executed by nuclear oestrogen (ER) and progesterone

Table 1 | Genes implicated in human uterine receptivity for implantation by data from independent microarray analysis

Comparison of five independent studies

Gene Molecule encoded (Putative function) LH+(8–10) vs

LH–(4–6)

LH+(7–9) vs

LH+(2–4)

LH+7 vs

LH+2

LH+(6–8) vs

LH–(3–5)

LH+8 vs

LH+3

Upregulated

ANXA4 Annexin-4 (SP) + + +

APOD Apolipoprotein D (LP) + + + +

BNIP2 BCL2/adenovirus E1B 19kDa interacting

protein-2 (cell-death protein)

+++

CLDN4 Claudin-4/CEP-R (R) + + +

C1R Complement component-1r (Imm) + + +

DAF* Decay accelerating factor for complement (Imm) + + + +

DF Complement factor D/Adipsin (Imm) + + +

DKK1 Dickkopf-1 (WNT antagonist) + + + +

GADD45A Growth arrest and DNA-damage-inducible

protein (DNA excision repair, cell-cycle regulator)

++++

GBP3 Guanylate-binding protein-2 (GTP-BP) + + +

ID4 Inhibitor DNA binding-4 (transcription

coregulator)

+++

IL15 Interleukin-15 (cytokine) + + + +

MAP3K5 Mitogen activated protein kinase kinase kinase 5

(MAPK signalling)

++++

MT1 Metallothionein-1 family proteins (MBP) + + + + +

MAOA Monoamine oxidase A (catechol-NT

metabolizing enzyme)

++++

PAEP Progestagen-associated endometrial protein

(SecP)

+++

SERPING1 Ser (or Cys), clade G (C1 inhibitor), member 1

(proteolysis inhibitor)

+++

SPP1

‡

Secreted phosphoprotein-1 (StrP) + + + + +

TGFB

TGFβ-super-family proteins

++ +

Downregulated

CCNB Cyclin B proteins (cell-cycle regulator) + + +

FRPHE Frizzled-related protein frpHE (WNT antagonist) + + +

GATA2 GATA-binding protein-2 + + +

MSX1 Hox Msh-like protein-1 + + +

MSX2 Hox Msh-like protein-2 + + +

OLFM1 Olfactomedin-related ER-localized protein-1

(SecP)

++++

Assuming a cycle length of 28–30 days, the surge in the levels of luteinizing hormone (LH) at mid-cycle heralds the onset of ovulation (BOX 2). The comparative

levels of global gene expression in the human uterus

presented here show that only few genes reveal similar changes (up or downregulation) across five

experiments. This poor consistency is perhaps due to changes in the timing of the assay, experimental designs, methods for data analysis, and/or geographical

location where subjects reside or the geographical location of the origin of subjects selected. The experiments compare gene-expression profiles between post-LH (+)

surge versus pre-LH surge (–) or early versus late post-LH surge. The receptive period in humans spans the period from days 7–10 after the LH surge (LH+(7–10)).

Genetic studies in mice might prove fruitful to assess whether these genes are critical to uterine receptivity in humans, since such studies are not possible in

humans except for the identification of mutations of these genes in human populations. *DAF, also known as CD55, Cromer blood-group system.

‡

SPP1, also known

as osteopontin. BP, binding protein; GF, growth factor; Hox, homeobox; Imm, immunomodulator; LP, lipoprotein; MBP, metal-binding protein; NT, neurotransmitter;

R, receptor; SecP, secretory protein; SP, signalling protein; StrP, structural protein; TF, transcription factor.

REVIEWS

190

MARCH 2006

VOLUME 7 www.nature.com/reviews/genetics

(PR) receptors. The recent discovery of ER (ERα and

ERβ) and PR (PRA and PRB) isoforms and studies of

the effect of their selective deletion provide evidence

for their isoform-specific functions in uterine biology

and implantation.

–/–

uteri are hypoplastic and unable

to support implantation

, whereas Er

–/–

uteri retain

biological functions that allow normal implantation

Interestingly, P

is sufficient for decidualization in Er

–/–

mice in response to artificial stimuli, which indicates

that ERα might be essential for blastocyst

attachment,

but dispensable for subsequent decidualization

43,44

. The

uterus expresses

PRA and PRB. While mice that lack both

PRA and PRB show many defects

in ovarian and uter-

ine functions, which leads to female infertility

, these

responses are normal in mice that are missing only PRB,

which indicates that essential P

-regulated functions are

primarily mediated by PRA

Cytokines. Among the cytokines, LIF, a member of the

interleukin-6 (IL-6) family, is crucial for uterine prepara-

tion for implantation. It binds to the LIF receptor and

gp130 as a common signal-transduction partner

with other cytokines. In mice,

Lif is expressed first in uter-

ine glands on the morning of day 4, and then in stromal

cells that surround the blastocyst during attachment

47,48

This indicates that LIF has a dual role: initially in uterine

preparation and later in attachment. Blastocysts remain

‘dormant’ in Lif

–/–

mice and do not implant, an effect

that depends on the uterine LIF mutant status

47,48

. The

molecular mechanism by which LIF executes its effects

on implantation is still unclear, although inactivation

of the gp130 protein by deleting its signal transducer

and activator of transcription (STAT) binding sites also

results in implantation failure

. Uterine Lif expression is

high around the time of implantation in other species,

including humans

Homeobox proteins. Several homeobox transcription

factors are crucial to uterine receptivity and implan-

tation. In mice, two Abdominal-B-like Hox genes,

Hoxa10 and Hoxa11, are expressed in uterine stromal

cells during the receptive phase. This expression per-

sists during postimplantation decidualization, which

might indicates an overlapping role for the two genes

in uterine receptivity, implantation and decidualiza-

tion

50–53

. Most Hoxa10

–/–

females are infertile, primarily

due to a reduced stromal-cell proliferation and the

consequent failure to decidualize

50,52

. However, Hoxa10

does not seem crucial for uterine receptivity, because

initial uterine attachment of blastocysts can occur in

Hoxa10

–/–

mice, and Hoxa10 expression is normal in

Lif

–/–

uteri

. In contrast, Hoxa11

–/–

uteri are hypoplastic,

have fewer glands and show a more severe phenotype

than Hoxa10

–/–

mice

. More importantly, the absence

of Lif expression in Hoxa11

–/–

uteri indicates that

Hoxa11 might be crucial to uterine receptivity and later

events of implantation

. Both Hoxa10 and Hoxa11 are

upregulated in the human uterus during the secretory

phase, which indicates that they might have a role in

uterine receptivity

. Gene-targeting experiments show

that blastocysts fail to implant in

Hmx3

–/–

mice, but the

reason for this failure remains unknown because Hmx3,

which belongs to a different homeobox gene family to

Hox genes, is mainly expressed in the

myometrium

Another homeobox gene,

Msx1, is transiently

expressed in the mouse uterine epithelium during the

receptive period, but disappears at the time of blastocyst

attachment or when the uterus enters the nonreceptive

phase

. Sustained expression of Msx1 in Lif

–/–

mice fur-

ther reinforces the importance of Msx1 in uterine recep-

tivity. It is interesting that Msx1 is downregulated in the

receptive human

endometrium

58–62

(TABLE 1). However, a

definitive role for Msx1 in uterine receptivity requires

conditional uterine deletion, because offspring that are

missing Msx1 die shortly after birth due to craniofacial

defects

Morphogens. One less-explored area is the role of

morphogens in uterine receptivity and implantation.

Embryo–uterine interactions during implantation

share many features with reciprocal epithelial–

mesenchymal interactions during embryogenesis, and

both involve evolutionarily conserved signalling path-

ways. The importance of hedgehog (HH), WNT and

bone-morphogenetic-protein (BMP) signalling in uter-

ine receptivity was recently explored. The genes encoding

the components of the HH signalling pathway, namely

Indian hedgehog (

Ihh), HH-binding protein/receptor

Patched (Ptc) and the transcription factors Gli1–3 are

expressed in the mouse uterus

64,65

. Ihh expression is

-dependent and reaches high levels in epithelial cells

on day 4, while that of Ptc, Gli1 and Gli2 is upregulated in

the underlying stroma. In day 4 uterine-explant cultures,

recombinant N-sonic hedgehog (N-SHH) stimulates

mesenchymal-cell proliferation, a characteristic of the

receptive phase

. These findings indicate that epithelial

IHH functions as a paracrine growth factor for stromal

cells and that this epithelial–mesenchymal signalling is

important for uterine receptivity.

The roles of WNT and BMP signalling in preserv-

ing tissue boundaries in the adult uterus remain largely

unknown.

sfrp4, a WNT antagonist and a member of the

secreted Frizzled-related proteins (sFRPs), and

Noggin,

an anti-BMP, are expressed in the uterine stroma during

the receptive phase

. Wnt4 and Bmp2 are not expressed

at this time, but are induced in the stroma with the onset

of blastocyst attachment, and thereafter with disappear-

ing expression of the antagonists

54,66

. These findings

indicate that while HH signalling participates in uterine

receptivity, WNT4 and BMP2 are involved in the attach-

ment reaction and postimplantation events.

Why, then, are sfrp4 and Noggin expressed in the

absence of their ligands? Do they have functions that

are independent of their ligands? Are other members

of the ligand family expressed in the uterus? A marked

downregulation of sfrp4 in Lif

–/–

uteri indicates that

a WNT-signalling component is important in uter-

ine preparation

. Alternatively, this downregulation

might be a consequence of compromised uterine func-

tion in the absence of Lif. Of the WNT family,

Wnt7a

is expressed in the LE in adult females, and deletion of

the Wnt7a gene shows global posterior shifting of the

Hypoplastic

Refers to an underdeveloped

tissue or organ.

Decidualization

Transformation of stromal cells

into morphologically and

functionally distinct cells. Part

of decidualized tissue is shed

at parturition.

Attachment

A process by which the

blastocyst trophectoderm is

brought into physical and

physiological contact with the

uterine luminal epithelium.

Myometrium

The muscular outer layer of

the uterus, which is comprised

of longitudinal and circular

muscle fibers.

Endometrium

The inner lining of the uterus;

it is primarily comprised of

stromal cells (the supporting

tissue of an organ) and

epithelial cells of both luminal

and glandular types. Part of the

endometrium is shed during

menstruation.

REVIEWS

NATURE REVIEWS

GENETICS VOLUME 7

MARCH 2006

191

Table 2 | Genes critical to uterine biology and implantation: results of mouse knockout models

Genes Molecule encoded (Putative function) HomoloGene

No. (NCBI)

Knockout phenotype in females Refs

Uterine patterning during postnatal growth

Hoxa10 Homeobox A10 (TF) 7365 Homeotic transformation of anterior uterus to oviduct 50,53,

114

Hoxa11 Homeobox A11 (TF) 4033 No uterine glands; partial homeotic transformation of

uterus to oviduct

Wnt5a WNT-5a protein (SP) 20720 No morphologically

defined cervix; no uterine glands 115

Wnt7a WNT-7a protein (SP) 20969 Abnormal oviduct and uterine development*; infertility 67

Uterine physiology in adult life

Adamts1 A disintegrin-like and metalloprotease with

thrombospondin-type-1 motif-1 (Enzyme, tissue

remodelling

21381 Impaired follicular development and fertilization;

uterine cysts; subfertility

116

Bteb1 Basic transcription-element-binding protein-1 (TF) 931, 79195 Uterine hypoplasia; compromised uterine P

function;

impaired embryo implantation; subfertility (ME)

117

Cenpb Centromere protein B (Centromere assembly) 1370 Disrupted luminal and glandular uterine epithelia;

subfertility (genetic-background-dependent)

118

Cyp27b1

25-hydroxyvitamin D 1α–hydroxylase enzyme

(Vitamin D metabolism)

37139 Uterine hypoplasia; absence of corpus luteum;

infertility

119

Esr1

Oestrogen receptor-α (NR,TF)

47906 Ovarian cysts; uterine hypoplasia; infertility 42

Igf1 Insulin-like growth factor-1 (GF) 515 Ovulation failure; uterine myometrial hypoplasia;

infertility

120

Pgr Progesterone receptor (NR,TF) 713 Unopposed oestrogen action; uterine hyperplasia;

infertility

Ube3a

‡

Ubiquitin-protein ligase E3A (Protein modification,

proteolysis and peptidolysis)

7988 Impaired follicular development and uterine

hypoplasia; subfertility

121

Vdr Vitamin D receptor (R,TF) 37297 Uterine hypoplasia with impaired folliculogenesis;

infertility

122

Uterine preparation for initiating implantation

Bsg Basigin (Immunoglobulin) 1308, 45225 Defective fertilization; no implantation 123,124

Esr1

Oestrogen receptor-α (NR,TF)

47906 No uterine attachment, but uterine responsiveness to

decidualization persists with P

priming

42,44

Fkbp52 FK506-binding protein-4 (Immunophilin co-

chaperone for steroid hormone NRs)

36085, 43060 Compromised P

function; no uterine receptivity (ME) 69

Gp130/

Stat

GP130/Signal Ttransducer and activator of

transcription (Cytokine-receptor signalling)

1645 No implantation (ME) 49

Hmx3 H6 homeobox-3 (TF) 40612 No implantation (ME) 57

LpA3 Lysophosphatidic acid receptor-3 (LPA signalling) 8123 Deferred, on-time implantation; aberrant embryo

spacing; postimplantation defects; small litter size (ME)

Lif Leukaemia inhibitory factor (Cytokine) 1734 No implantation (ME) 48

Pgr Progesterone receptor (NR,TF) 713 No implantation or decidualization (ME) 45

Pla2g4a Phospholipase A2, group IVA

(Arachidonic-acid-

releasing enzyme)

32059 Deferred on-time implantation; aberrant embryo

spacing; postimplantation defects; small litter size (ME)

Ppard

Peroxisome proliferator-activated receptor-δ

(NR,TF)

4544 4–6 h delay in initiating embryo attachment; placental

defects; subfertility

76,125

Ptgs2

Prostaglandin-endoperoxide synthase-2

(Prostaglandin synthesis)

31000 Multiple reproductive failures, including defective

attachment reaction; genetic-background-dependent

75,77

Uterine decidualization

Fkbp52 FK506-binding protein-4 (Immunophilin co-

chaperone for steroid hormone NRs)

36085, 43060 Compromised P

function; defective decidualization

(ME)

Hoxa10 Homeobox A10 (TF) 7365 Defective decidualization; reduced fertility (ME) 50,52,53

Hoxa11 Homeobox A11 (TF) 4033 Defective implantation and decidualization; infertility 51

Il11ra1

Interleukin-11 receptor-α1 (Cytokine signalling)

3316 Impaired decidualization; infertility 126,127

Pgr Progesterone receptor (NR,TF) 713 Lack of decidual response even after P

priming 45

Ptgs2

Prostaglandin-endoperoxide synthase-2

(Prostaglandin synthesis)

31000 Defective decidualization; reduced angiogenic

response

75,85

*Also, reduced stromal tissue, lack

of uterine glands and disorganized myometrium.

‡

Ube3a, also known as E6AP ubiquitin-protein ligase.

Cytosolic, calcium-

dependent.

Also known as cyclooxygenase-2 (COX2). GF, growth factor; ME, maternal effect; NR, nuclear receptor; R, receptor, SP, signalling protein; TF,

transcription factor.

REVIEWS

192

MARCH 2006

VOLUME 7 www.nature.com/reviews/genetics

Blastocyst

activation

HB-EGF

ErbB

PRA

LPA3

Adhesion

molecules

ErbB1/4 ↑

CB1 ↓

MYO

ERα

COX2

cPLA2α

PPARδ

WNT4

BMP2

LIF

FKBP52

LIF

AttachmentApposition

Postimplantation

Bmp7; Dan; Crim1

Fgf2; sFRP4

Noggin; Fgf10

Wnt4

Bmp2

ICM

Basal lamina

A thin sheet of proteoglycans

and glycoproteins that are

secreted by cells as an

extracellular matrix. It is also

called the basement membrane

and influences cell polarity,

differentiation and migration.

Decidual cells

In the mouse, the cells that

surround the implanting

blastocyst.

Oedema

Fluid accumulation in the

intercellular tissue spaces.

Luminal closure

The closure of the uterine

lumen, resulting in closer

contact between the luminal

epithelial linings; this step is

essential for blastocyst

attachment.

reproductive tract, with the loss of Hoxa10 and Hoxa11.

Wnt7a

–/–

females are infertile, with uteri that lack glands

and disorganized myometria, which indicates that Wnt7a

might be crucial for normal uterine cellular architec-

ture

. Of the Bmp genes that have been studied, Bmp4–7

and 8a do not show the same highly localized expression

pattern as Bmp2 during attachment

. An investigation

that spans other members of the WNT and BMP fami-

lies, their receptors and putative antagonists is warranted

to better understand the roles of these morphogens in

uterine receptivity.

Signalling during implantation

The process of implantation is classified into three

stages: apposition, attachment (adhesion) and penetra-

tion. During apposition, the Tr becomes closely apposed

to the LE. This is followed by the attachment stage, when

the association of the Tr and LE is sufficiently intimate to

resist dislodging of the blastocyst by flushing the uterine

lumen. The first sign of the attachment reaction occurs

on the evening of day 4 in mice, and coincides with a

localized increase in stromal vascular permeability at

the site of blastocyst attachment. Penetration involves

invasion of the embryo through the LE and

basal lamina

into the stroma, to establish a vascular relationship with

the mother. At this stage, stromal-cell differentiation into

decidual cells (decidualization) is extensive and leads to

the loss of the LE at the site of the implanting blastocyst.

(Note, however, that stromal-cell decidualization also

occurs in women during the luteal phase of the men-

strual cycle, in the absence of an embryo.) The dynamic

and overlapping expression of signalling molecules dur-

ing these three stages makes it difficult to assign the con-

tribution of specific signalling pathways to a particular

stage (

FIG. 2; TABLE 2).

Apposition. In rodents, a generalized stromal

oedema

leads to uterine

luminal closure, resulting in interdigita-

tion of microvilli of the Tr and LE (apposition). Luminal

closure occurs in pregnant or pseudopregnant uteri, and

therefore does not require the presence of blastocysts. P

priming, however, is essential for closure. This is sup-

ported by the absence of luminal closure in pregnant mice

that are missing FK506 binding protein-4 (FKBP52), a

co-chaperone that is required for appropriate uterine

PR function

68,69

. Fkbp52 expression overlaps with that

of PR in the stroma before the attachment reaction,

and Fkbp52

–/–

females show implantation failure and

Figure 2 | Gene products participating in embryo implantation. a | Signalling pathways that are known to coordinate

blastocyst apposition and attachment in the mouse uterus. Apposition and attachment are key steps in implantation and

absolutely depend on the synchronized development of the blastocyst to implantation competency and differentiation

of the uterus to the receptive stage. Ovarian oestrogen and progesterone, acting through their cognate nuclear

receptors, influence several locally produced growth factors, adhesion molecules, cytokines, transcription factors and

vasoactive mediators and their receptors in the uterus and/or blastocyst to coordinate blastocyst–uterine crosstalk. This

crosstalk further influences some of the signalling pathways to ensure the successful execution of the implantation

process. b | Region-specific expression patterns of morphogens in the mouse deciduum during the postimplantation

period. This scheme is based on in situ hybridization of the indicated genes in a representative cross-section of an

implantation chamber on day 7 of pregnancy. AM, antimesometrial pole; BMP2, bone morphogenetic protein-2; CB1,

brain-type cannabinoid receptor-1; COX2, cyclooxygenase-2; cPLA2α, cytosolic phospholipase A

2α

; Crim1, cysteine-rich

transmembrane BMP-regulator-1; Dan, differential screening-selected gene aberrative in neuroblastoma; Em, embryo;

ERα, nuclear oestrogen receptor-α; ErbB, EGF-receptor family; FGF, fibroblast growth factor; FKBP52, FK506 binding

protein-4; GE, glandular epithelium; HB-EGF, heparin-binding EGF-like growth factor; ICM, inner cell mass; LE, luminal

epithelium; LIF, leukaemia inhibitory factor; LPA3, lysophosphatidic-acid receptor-3; M, mesometrial pole; MYO,

myometrium; PPARδ, peroxisome-proliferator-activated receptor-δ; PRA; nuclear progesterone receptor A; S, stroma;

sFRP4, secreted Frizzled-related protein-4; Tr, trophectoderm; V, blood vessels; ZP, zona pellucida.

REVIEWS

NATURE REVIEWS

GENETICS VOLUME 7

MARCH 2006

193

Integrins

A family of receptors for

various extracellular-matrix

ligands that modulate cell–cell

adhesion and signal

transduction. Each integrin has

two subunits, α and β, and

each αβ combination has a

unique binding specificity and

unique signalling properties.

Selectins

A group of cell-adhesion

molecules, including L-selectin,

E-selectin and P-selectin, that

bind to carbohydrates.

Galectins

A family of lectins with

galactose-binding ability.

Trophinin–tastin–bystin

complex

A homophilic cell-adhesion

complex that is comprised of

membrane–cytoplasmic

proteins.

Prostaglandins

(PG). Vasoactive lipid mediators

that are implicated in various

pathophysiological processes,

including vascular permeability,

angiogenesis and cell migration.

downregulation of the P

-responsive genes Areg (which

encodes amphiregulin), Hoxa10 and Ihh in the uterus.

However, although P

priming via PR is essential for

luminal closure and apposition, blastocyst attachment

cannot occur unless the P

-primed uterus is exposed

to oestrogen.

The signalling pathway that is initiated by HB-EGF has

been studied extensively during apposition and attach-

ment because HB-EGF is an early molecular marker of

embryo–uterine crosstalk

. Heg f1 is expressed in the

mouse LE at the site of blastocyst apposition several hours

before attachment, and this persists through the early

attachment phase. HB-EGF is produced as soluble and

transmembrane forms. Molecular and genetic evidence

show that it influences embryonic functions as a para-

crine and/or juxtacrine factor by interacting with ErbB1

and/or ErbB4, which are expressed on the blastocyst cell

surface

30,31,38

. Most Hegf1

–/–

mice die during prenatal and

early postnatal life due to cardiac defects

, precluding an

examination of the implantation phenotype.

Implantation-competent blastocysts that also express

Hegf1 induce expression of the gene in the uterus in a

paracrine manner

. This auto-induction loop is per-

haps the first example of molecular crosstalk between

the blastocyst and uterus, initiating the attachment reac-

tion. HB-EGF also has a role in human implantation. Its

expression is maximal in the receptive endometrium,

and cells that express transmembrane HB-EGF adhere

to blastocysts that display cell-surface ErbB4

(REF. 72).

Attachment. It is correctly assumed that adhesive-

signalling systems are required for the attachment phase.

Indeed, numerous glycoproteins and carbohydrate

ligands and their receptors are expressed in LE and

Tr cells around the time of implantation. The most

important adhesion molecules that are implicated in

this process are

integrins, selectins, galectins, heparan

sulfate proteoglycans (HSPGs), mucin-1, cadherins

and the

trophinin–tastin–bystin complex

1,2,4

. Integrins

and selectins are of special interest because of their

unique functional features. In the human uterus,

integrin αvβ3 is localized to the LE during the recep-

tive phase, and its aberrant expression is correlated

with infertility and recurrent pregnancy loss

. Recent

evidence shows that selectin signalling is also important

in human implantation. While selectin oligosaccharide

ligands are expressed in the receptive LE,

L-selectin

molecules are displayed on the Tr cell surface

. More

importantly, beads that are coated with specific selectin

ligands adhere to trophoblast cells and, conversely, iso-

lated trophoblast cells bind preferentially to the recep-

tive uterine surface. These findings indicate that the

selectin-adhesion system constitutes an initial step

in human implantation. However, apparently normal

fertility in mice that lack L-selectin indicates a species-

specific variation in the adhesion cascade during implan-

tation. As stated before, Lif also seems to be important

for the attachment process, because Lif

–/–

mice show a

lack of HB-EGF and aberrant cyclooxygenase-2 (

Cox2)

expression in blastocysts during the anticipated time

of attachment

47,74

Penetration. One key event in implantation is an

increased endometrial vascular permeability at the

site of blastocyst attachment and penetration

(FIG. 2),

a process that involves the action of

prostaglandins

(PGs). COX1 and COX2 mediate PG synthesis and

are encoded by Ptgs1 and Ptgs2, respectively. Ptgs2

expression is unique in the mouse uterus, and shows

expression in the LE and underlying stromal cells at

the site of blastocyst attachment

. It is speculated that

HB-EGF that is produced in the uterus and embryo

induces uterine Ptgs2 expression. Ptgs2

–/–

females are

largely infertile, with defective ovulation, fertiliza-

tion, implantation and decidualization

. COX2-

derived prostacyclin (PGI

) is the primary PG that is

produced at the implantation site, and implantation

defects are improved in Ptgs2

–/–

mice by PG admin-

istration

. Evidence indicates that PGI

participates

in implantation via the activation of peroxisome-

proliferator-activated receptor-δ (

PPARδ), the expression

of which overlaps with Ptgs2 at the implantation site

However, depending on the genetic background,

COX1 can compensate for COX2 to improve infertil-

ity in Ptgs2

–/–

females

. Cox2 is also expressed in the

uterus and/or blastocyst during implantation in several

species, including primates

, indicating a conserved

function for COX2 in implantation.

The function of PG is further illustrated by the

reduced fertility of mice that lack cytoplasmic phos-

pholipase A

2α

(cPLA2α), which generates a precursor

for PG synthesis. Compromised fertility is due to the

deferral of on-time implantation, which leads to inap-

propriate embryo spacing, retarded feto-placental

development and reduced litter size

. These results

reveal that the cPLA2α–COX2 signalling axis is crucial

to implantation. Signalling by lysophosphatidic acid

(LPA), which belongs to a lysophospholipid group, also

influences blastocyst attachment in mice by activating

the G-protein-coupled receptor LPA3

(REF. 80). Like the

cPla2

–/–

mice, lpA3

–/–

females show deferred implanta-

tion and its associated defects. The treatment of both

cPla2

–/–

and lpA3

–/–

mice with PGs resumes on-time

implantation, but embryo crowding persists. Phenotypic

similarities between lpA3-

and cPla2

-deficient mice

and reduced levels of uterine COX2 in lpA3

–/–

mice

identify COX2 as a common signalling pathway.

Implications for human fertility. From the results

discussed above, one important finding is that a short

delay in blastocyst attachment creates an adverse

ripple effect throughout the course of pregnancy,

which leads to defective feto–placental development

and poor pregnancy outcome. This indicates a new

concept in which embryo–uterine interactions dur-

ing implantation set up subsequent developmental

programming. This idea is supported by the clinical

finding that implantation beyond the normal window

of receptivity is associated with a higher risk of early

pregnancy loss in women

. The downstream pathways

of PG signalling that participate in the ripple effect

remain unknown. In light of these findings, one can

assume that many previous studies that describe early

REVIEWS

194

MARCH 2006

VOLUME 7 www.nature.com/reviews/genetics

Placenta previa

A condition in humans in which

the placenta is situated close

to or covering the cervix.

Hemochorial placentation

The process by which maternal

blood comes in direct contact

with the trophoblast.

or mid-gestational embryonic lethality arising from

specific mutations might have originated at the time

of implantation.

Another unresolved issue is embryo spacing in the

uterus. While the LPA3–cPLA2α–COX2

signalling axis

is important for normal embryo spacing, attachment and

penetration, no molecule has been found to rescue the

spacing defect in mice that are mutant for the processes

involved. Answers to the question of how embryo spac-

ing is regulated might provide insights into the aetiology

placenta previa in humans. Is it possible that embryo

spacing is regulated by local factors that are associated

with PG signalling? BMPs are required for the spacing

of tissue structures during development

, and local

delivery of BMP2 or BMP4 in the uterus causes aberrant

embryo spacing

. There is also genetic evidence that

BMP5 and NODAL are important for this process

. As

there is a relationship between BMP and PG signalling

in other systems

, these pathways might work together

to influence embryo spacing.

Postimplantation uterine development

Uterine stromal cells that surround the blastocyst

undergo decidualization following attachment, even-

tually embedding the embryo in the antimesometrial

stromal bed. One function of the deciduum is to provide

nutritional support to the developing embryo before

the establishment of a functional placenta. Numerous

signalling molecules, including cytokines, homeobox

transcription factors, cell-cycle molecules, extracellular-

matrix remodelling factors and lipid mediators, are

expressed in the endometrium during decidualization

and are crucial to this process

. Here, we focus on the

less-explored areas, such as uterine angiogenesis and

establishment of the uterine–embryonic axis during the

postimplantation period

(FIG. 2b).

Uterine angiogenesis. Under physiological conditions

in adult females, angiogenesis primarily occurs in the

uterus and ovary during the reproductive cycle and preg-

nancy. Angiogenesis is essential to normal implantation

and placentation, and is profoundly influenced by vas-

cular endothelial growth factor (

VEGF) and angiopoi-

etins. PGs, because of their role in angiogenesis in other

systems, are also thought to participate in uterine angio-

genesis during pregnancy. But what is the link between

VEGF, angiopoietin and PG signalling?

The VEGF

receptor FLK1 (also known as KDR, kinase-insert-

domain protein receptor) is a marker of endothelial

cells during angiogenesis. Using Ptgs2

–/–

x Flk1

+/–LacZ

reporter mice, it was shown that COX2-derived PGs

markedly influence uterine angiogenesis during

decidualization by differentially regulating VEGF and

angiopoietin signalling cascades

. Uterine angiogen-

esis in Ptgs2

–/–

mice is severely compromised, owing to

defective VEGF, but not angiopoietin, signalling and

this defect is rescued by exogenous PG. Because PGs

coordinate VEGF signalling with that of angiopoietins

during decidual angiogenesis, one cause of compromised

implantation and decidualization in Ptgs2

–/–

mice could

be dysregulated vascular events.

Establishment of the uterine–embryonic axis. The

adult uterus undergoes dynamic cellular and molecu-

lar changes during pregnancy, but how these changes

are coordinated to specify the allocation of new cell

types, for example, decidual cells and their boundaries,

remains largely unknown. Decidualization is initiated

at the antimesometrial pole, subsequently extend-

ing to the mesometrial pole, the presumptive site of

placentation. This orients the implantation chamber

in an antimesometrial–mesometrial (AM–M) direc-

tion, in alignment with the embryonic axis. It is still

unclear how the implantation chamber is oriented

and grows in an AM–M direction, with the decidual

reaction spreading in the same direction. It is also not

known how decidual cell growth is restricted, leaving a

layer of undifferentiated stromal cells underneath the

myometrium.

It is speculated that WNT signalling, in collabora-

tion with those of BMP and fibroblast growth factor

(FGF), helps to orient the implantation chamber in

the AM–M direction and specifies these boundaries

during decidualization

(FIG. 2b); in particular, differen-

tial WNT4 signalling seems to participate in making

this boundary

. An inverse relationship with respect

to Bmp2 and Noggin expression that is observed dur-

ing implantation and decidualization also indicates

differential BMP signalling during early pregnancy

However, the expression of

Dan (differential screen-

ing-selected gene aberrative in neuroblastoma), a

member of the Dan/Dante Bmp-antagonist gene family,

and

Crim1 (cysteine-rich transmembrane BMP-

regulator-1), which encodes a protein that is thought to

bind BMP, partially overlap with that of Bmp2 expres-

sion. Furthermore, antimesometrial expression of

Fgf2,

in contrast to mesometrial expression of

Fgf10, adds to

evidence that the AM–M orientation of the uterus dur-

ing early pregnancy is influenced by differential gene

expression

. We speculate that uterine orientation helps

to establish embryonic orientation during development,

and that the failure of the implantation chamber to

orient itself in an AM–M direction is likely to disrupt

embryonic orientation. Therefore, these developmental

genes are not only important for establishing bound-

aries and polarities during embryogenesis, but also for

establishing the orientation of the growing implantation

chamber and creating boundaries to prevent undifferen-

tiated stromal cells from decidualizing (the undifferen-

tiated stromal cells might serve to replenish the stroma

after parturition).

How mice can help humans

Studies in mice have provided insights into the molecu-

lar basis of human implantation because of their shared

features. Both mouse and human embryos can develop

in vitro in simple, defined media. In both species,

embryo implantation leads to stromal decidualization

— embryos embed in the antimesometrial stroma and

placentation is

hemochorial.

Mouse embryos grow more effectively when they

are cultured in a small volume

. This protocol, which is

practiced by some clinics, has shown improved embryo

REVIEWS

NATURE REVIEWS

GENETICS VOLUME 7

MARCH 2006

195

MALDI mass spectrometry

(Matrix-assisted laser

desorption/ionization mass

spectrometry). It is based on

the co-crystallization of a test

compound with an ultraviolet-

light-absorbing matrix, which

allows ionization using laser

excitation to determine the

mass of the test compound.

Preeclampsia

The development of

hypertension with proteinuria

(excess protein in urine) and/or

oedema during pregnancy;

early onset occurs from

defective trophoblast function.

development in human IVF programs. However, the

pregnancy success rate remains poor (~30%) due to

the transfer of IVF-derived embryos into nonrecep-

tive uteri. One cause of nonreceptivity might be high

oestrogen levels; this results from the gonadotropin

treatment that is given to women to stimulate the

ovaries to produce multiple eggs. Indeed, the range of

oestrogen levels that determines uterine receptivity in

mice is narrow

. This remarkable uterine sensitivity

to oestrogen might be important to ensure the correct

timing of implantation, which is crucial to pregnancy

outcome. Understanding the cause of uterine non-

receptivity at higher oestrogen levels might make it

possible to extend uterine receptivity by using an aro-

matase inhibitor to neutralize excess oestrogen during

gonadotropin stimulation.

Early onset of intrauterine growth restriction, recur-

rent abortion,

preeclampsia and preterm delivery are

important reproductive health issues, and are associated

with placental deficiencies. A transient postponement of

blastocyst attachment in mice produces a detrimental

ripple effect throughout pregnancy, which indicates that

these end results are due to defective implantation. Such

defects could be corrected, because signalling by LIF,

HB-EGF, COX2 and HOX family members, which are

important at different stages of implantation in mice, are

also thought to be important for human implantation. In

fact, downregulation of HB-EGF expression in the human

trophoblast is associated with preeclampsia

. Further

insights into these and the recently identified pathways

that are described above might improve pregnancy

success and could also help in designing new and improved

contraceptives. There is a need to develop nonsteroidal

contraceptives so that women are spared the complica-

tions of hormonal imbalances and the risk of developing

gynecological cancers. Molecular approaches to disrupt

LIF–STAT, FKBP52 or LPA3–COX2 signalling pathways

might be considered for potential contraceptives.

Conclusion

Implantation is an incredibly useful biological system,

a better understanding of which will advance our

know ledge in several basic physiological processes.

These include: the paracrine and juxtacrine epithelial–

epithelial interactions that occur between the Tr and LE

during attachment; the epithelial–mesenchymal interac-

tion between the LE and the stroma; Tr–epithelium–stroma

interactions, involving cell migration and invasion; vas-

cular permeability and adult angiogenesis; and regulated

growth (proliferation, differentiation, polyploidy and

apoptosis) during stromal decidualization. Despite the

large strides that have been made by applying genom-

ics and proteomic approaches to rodents, the field faces

many important challenges

(BOX 4).

Implantation involves numerous signalling pathways

that are common to other systems under either normal or

pathological conditions. Therefore, research on implanta-

tion should appeal to a broader range of scientists, not

solely to reproductive or developmental biologists.

For

example, many of the features and signalling pathways

that are required during implantation are also active

during tumourigenesis — the difference being that tight

Box 4 | Future challenges for reproductive biology and reproductive medicine

• There is a need to identify reliable markers of uterine receptivity and to develop the means to extend uterine receptivity

or treat nonreceptivity to improve the pregnancy rate in in vitro fertilization and embryo-transfer programmes.

Overcoming these challenges will lessen the need to transfer multiple embryos to increase the pregnancy rate and the

resulting complications of multiple pregnancies.

• Although various signalling pathways operate during implantation, it is still unclear whether they work independently, in

parallel or converge on a common pathway.

• Suitable animal models must continue to be developed to define the molecular communication between the uterus and

embryo. Such studies require information about the contribution that is made by each of the two tissues, a task not easily

achievable in humans because of experimental difficulties and ethical restrictions on research with human embryos.

• Another challenge is to identify gene promoters for creating inducible Cre-transgenic mice for conditional gene deletion

specifically in uterine cells; genome-wide deletion of many implantation-associated genes leads to embryonic lethality,

precluding studies on implantation. This approach will also help to elucidate the long-term versus acute effects of a gene

during implantation. This is particularly important in the context of the adaptation of animals to a new make-up. For

example, deletion of one gene that does not affect pregnancy under normal conditions shows adverse effects under a

stress situation

109

• Efforts should continue in establishing a relevant in vitro model of implantation to study the hierarchy of events that are

triggered by the embryo, and the function of specific signalling molecules.

• There is a need to properly annotate the markers of uterine receptivity that are derived from microarray experiments in

rodents and humans

58–62,110,111

with results from functional analyses. Comparative proteomics between wild-type and

mutant uteri (an assay that is under-exploited in the field) should provide unique information

• Another approach that shows great promise is the direct analysis of tissue sections by MALDI mass spectrometry to

identify the spatial localization of proteins

112

. This approach is attractive for comparing proteome profiles of implantation

versus interimplantation sites or between different regions within an implantation site.

• The nature of embryonic signals that influence uterine functions is mostly unknown. In rodents and humans, the limiting

factor is the availability of adequate amounts of tissues for analysis. With the advent of microscale proteomics and

genomics, it might now be possible to identify embryonic signals during implantation. Once potential molecules are

identified, their functions could be assessed by local application into the uterus using blastocyst-sized beads as carriers

REVIEWS

196

MARCH 2006

VOLUME 7 www.nature.com/reviews/genetics

1. Carson, D. D. et al. Embryo implantation. Dev. Biol.

223, 217–237 (2000).

2. Dey, S. K. et al. Molecular cues to implantation.

Endocr. Rev. 25, 341–373 (2004).

3. Paria, B. C., Reese, J., Das, S. K. & Dey, S. K.

Deciphering the cross-talk of implantation: advances

and challenges. Science 296, 2185–2188 (2002).

4. Red-Horse, K. et al. Trophoblast differentiation during

embryo implantation and formation of the maternal-

fetal interface. J. Clin. Invest. 114, 744–754 (2004).

5. Enders, A. C. & Schlafke, S. A morphological analysis

of early implantation stages in the rat. Am. J. Anat.

120, 195–226 (1967).

6. Nothias, J. Y., Majumder, S., Kaneko, K. J. &

DePamphilis, M. L. Regulation of gene expression at

the beginning of mammalian development. J. Biol.

Chem. 270, 22077–22080 (1995).

7. Latham, K. E., Garrels, J. I., Chang, C. & Solter, D.

Quantitative analysis of protein synthesis in mouse

embryos. I. Extensive reprogramming at the one- and

two-cell stages. Development 11 2 , 921–932 (1991).

8. Shi, C. Z. et al. Protein databases for compacted

eight-cell and blastocyst-stage mouse embryos.

Mol. Reprod. Dev. 37, 34–47 (1994).

9. Zimmermann, J. W. & Schultz, R. M. Analysis of gene

expression in the preimplantation mouse embryo: use

of mRNA differential display. Proc. Natl Acad. Sci.

USA 91, 5456–5460 (1994).

10. Ko, M. S. et al. Large-scale cDNA analysis reveals

phased gene expression patterns during

preimplantation mouse development. Development

127, 1737–1749 (2000).

11. Hamatani, T., Carter, M. G., Sharov, A. A. & Ko, M. S.

Dynamics of global gene expression changes during

mouse preimplantation development. Dev. Cell 6,

117–131 (2004).

This study, together with the work described in

reference 12, shows that mouse preimplantation

embryo development is a dynamic molecular

process that is governed by waves of gene

expression.

12. Wang, Q. T. et al. A genome-wide study of gene

activity reveals developmental signaling pathways in

the preimplantation mouse embryo. Dev. Cell 6,

133–144 (2004).

13. Zeng, F., Baldwin, D. A. & Schultz, R. M. Transcript

profiling during preimplantation mouse development.

Dev. Biol. 272, 483–496 (2004).

14.

Tong, Z. B. et al. Mater, a maternal effect gene

required for early embryonic development in mice.

Nature Genet. 26, 267–268 (2000).

15. Johnson, M. H. & McConnell, J. M. Lineage allocation

and cell polarity during mouse embryogenesis.

Semin. Cell Dev. Biol. 15, 583–597 (2004).

16. Rossant, J. Lineage development and polar

asymmetries in the peri-implantation mouse

blastocyst. Semin. Cell Dev. Biol. 15, 573–581

(2004).

17. Nichols, J. et al. Formation of pluripotent stem cells

in the mammalian embryo depends on the POU

transcription factor Oct4. Cell 95, 379–391 (1998).

This study shows that expression of Oct4 in inside

cells of mouse preimplantation embryos is required

for the generation of pluripotent cells. The other

key molecules for cell-lineage differentiation during

preimplantation development are described in

references 18–24.

18. Avilion, A. A. et al. Multipotent cell lineages in early

mouse development depend on SOX2 function. Genes

Dev. 17, 126–140 (2003).

19. Niwa, H., Miyazaki, J. & Smith, A. G. Quantitative

expression of Oct-3/4 defines differentiation,

dedifferentiation or self-renewal of ES cells. Nature

Genet. 24, 372–376 (2000).

20. Mitsui, K. et al. The homeoprotein Nanog is required

for maintenance of pluripotency in mouse epiblast and

ES cells. Cell 11 3, 631–642 (2003).

21. Chambers, I. et al. Functional expression cloning of

Nanog, a pluripotency sustaining factor in embryonic

stem cells. Cell 11 3, 643–655 (2003).

22. Boyer, L. A. et al. Core transcriptional regulatory

circuitry in human embryonic stem cells. Cell 122,

947–956 (2005).

23. Strumpf, D. et al. Cdx2 is required for correct cell fate

specification and differentiation of trophectoderm in

the mouse blastocyst. Development 132, 2093–2102

(2005).

24. Russ, A. P. et al. Eomesodermin is required for mouse

trophoblast development and mesoderm formation.

Nature 404, 95–99 (2000).

25. Paria, B. C., Huet-Hudson, Y. M. & Dey, S. K.

Blastocyst’s state of activity determines the

‘‘window’’ of implantation in the receptive mouse

uterus. Proc. Natl Acad. Sci. USA 90, 10159–10162

(1993).

This study, which uses a delayed-implantation

mouse model, showed for the first time that

the receptive state of the uterus alone is not

sufficient for successful implantation, but that

blastocysts must also achieve implantation

competency. The differential roles of oestrogen

and catecholoestrogens in establishing the

window of implantation are highlighted in

references 26 and 32.

26. Ma, W. G., Song, H., Das, S. K., Paria, B. C. & Dey, S. K.

Estrogen is a critical determinant that specifies the

duration of the window of uterine receptivity for

implantation. Proc. Natl Acad. Sci. USA 100,

2963–2968 (2003).

27. Lopes, F. L., Desmarais, J. A. & Murphy, B. D.

Embryonic diapause and its regulation. Reproduction

128, 669–678 (2004).

28. Renfree, M. B. & Shaw, G. Diapause. Annu. Rev.

Physiol. 62, 353–375 (2000).

29. Hamatani, T. et al. Global gene expression analysis

identifies molecular pathways distinguishing blastocyst

dormancy and activation. Proc. Natl Acad. Sci. USA

101, 10326–10331 (2004).

This study analyses global gene expression in

dormant and activated mouse blastocysts,

providing evidence that gene-expression patterns

are distinct at these two different physiological

states of the embryo.

30. Paria, B. C., Das, S. K., Andrews, G. K. & Dey, S. K.

Expression of the epidermal growth factor receptor

gene is regulated in mouse blastocysts during delayed

implantation. Proc. Natl Acad. Sci. USA 90, 55–59

(1993).

31. Raab, G. et al. Mouse preimplantation blastocysts

adhere to cells expressing the transmembrane form of

heparin-binding EGF-like growth factor. Development

122, 637–645 (1996).

32. Paria, B. C. et al. Coordination of differential effects of

primary estrogen and catecholestrogen on two distinct

targets mediates embryo implantation in the mouse.

Endocrinology 139, 5235–5246 (1998).

33. Guo, Y. et al. N–acylphosphatidylethanolamine-

hydrolyzing phospholipase D is an important

determinant of uterine anandamide levels during

implantation. J. Biol. Chem. 280, 23429–23432

(2005).

34. Paria, B. C., Das, S. K. & Dey, S. K.

The preimplantation mouse embryo is a target for

cannabinoid ligand-receptor signaling. Proc. Natl

Acad. Sci. USA 92, 9460–9464 (1995).

This work provided the first evidence for the

presence of the G-protein-coupled cannabinoid

receptors CB1 and CB2 in preimplantation mouse

embryos. The differential roles of

endocannabinoids in embryo–uterine interactions

during implantation are further illustrated in

references 33 and 35–37.

35. Wang, H. et al. Aberrant cannabinoid signaling

impairs oviductal transport of embryos. Nature Med.

10, 1074–1080 (2004).

36. Wang, H. et al. Differential G protein-coupled

cannabinoid receptor signaling by anandamide directs

blastocyst activation for implantation. Proc. Natl

Acad. Sci. USA 100, 14914–14919 (2003).

37. Paria, B. C. et al. Dysregulated cannabinoid signaling

disrupts uterine receptivity for embryo implantation.

J. Biol. Chem. 276, 20523–20528 (2001).

38. Wang, J., Mayernik, L., Schultz, J. F. & Armant, D. R.

Acceleration of trophoblast differentiation by

heparin-binding EGF-like growth factor is dependent

on the stage-specific activation of calcium influx by

ErbB receptors in developing mouse blastocysts.

Development 127, 33–44 (2000).

39. Stachecki, J. J. & Armant, D. R. Transient release of

calcium from inositol 1,4,5-trisphosphate-specific

stores regulates mouse preimplantation development.

Development 122, 2485–2496 (1996).

40. Wang, Y. et al. Entire mitogen activated protein kinase

(MAPK) pathway is present in preimplantation mouse

embryos. Dev. Dyn. 231, 72–87 (2004).

41. Riley, J. K. et al. The PI3K/Akt pathway is present and

functional in the preimplantation mouse embryo.

Dev. Biol. 284, 377–386 (2005).

42. Lubahn, D. B. et al. Alteration of reproductive

function but not prenatal sexual development after

insertional disruption of the mouse estrogen receptor

gene. Proc. Natl Acad. Sci. USA 90, 11162–11166

(1993).

43. Curtis, S. W., Clark, J., Myers, P. & Korach, K. S.

Disruption of estrogen signaling does not prevent

progesterone action in the estrogen receptor α

knockout mouse uterus. Proc. Natl Acad. Sci. USA 96,

3646–3651 (1999).

44. Paria, B. C., Tan, J., Lubahn, D. B., Dey, S. K. & Das, S. K.

Uterine decidual response occurs in estrogen

receptor-α-deficient mice. Endocrinology 140,

2704–2710 (1999).

regulation occurs during implantation, while dysregu-

lation of the same pathways occurs in tumourigenesis.

Therefore, understanding the intricacies of implanta-

tion might help to better understand the complexities of

tumourigenesis, and might one day reveal that ‘life and

death are linked by a common thread’.

Note added in proof

The discoveries that CDX2 and OCT3/4 are crucial

for specifying Tr and ICM cells, respectively, in pre-

implantation embryos have now been expanded to

show their roles in stem-cell biology. A report shows

that embryos that are missing CDX2 develop to blas-

tocysts, but fail to implant because they lack Tr. These

blastocysts have ICMs and generate ES cells when

grown in culture

128

. Another recent report shows that

ES cells can be forced to differentiate to trophoblast

stem (TS) cells by repressing OCT3/4

(REF. 129). This

study shows that CDX2 is dispensable for Tr differen-

tiation, but is required for TS-cell self renewal, which

indicates that the reciprocal inhibition of transcription

factors OCT3/4 and CDX2 participates in lineage dif-

ferentiation in mammalian embryos

129

. Although the

cell-lineage specification in mammalian embryos is

primarily thought to occur between the 8-cell and

blastocyst stages, the question of how embryonic

polarity is established is still a subject of debate. Gore

et al.

130

now show that maternal Squint, a mammalian

NODAL-related morphogen, localizes to two blast-

omeres at the 4-cell stage and specifies the dorsal axis,

which indicates that NODAL could also be involved in

determining polarity in early mammalian embryos.

REVIEWS

NATURE REVIEWS

GENETICS VOLUME 7

MARCH 2006

197

45. Lydon, J. P. et al. Mice lacking progesterone receptor

exhibit pleiotropic reproductive abnormalities. Genes

Dev. 9, 2266–2278 (1995).

46. Mulac-Jericevic, B., Mullinax, R. A., DeMayo, F. J.,

Lydon, J. P. & Conneely, O. M. Subgroup of

reproductive functions of progesterone mediated by

progesterone receptor-B-isoform. Science 289,

1751–1754 (2000).

47. Song, H., Lim, H., Das, S. K., Paria, B. C. & Dey, S. K.

Dysregulation of EGF family of growth factors and

COX-2 in the uterus during the preattachment and

attachment reactions of the blastocyst with the

luminal epithelium correlates with implantation failure

in LIF-deficient mice. Mol. Endocrinol. 14, 1147–1161

(2000).

48. Stewart, C. L. et al. Blastocyst implantation depends

on maternal expression of leukaemia inhibitory factor.

Nature 359, 76–79 (1992).

This study provided the first evidence that Lif is

expressed in mouse uterine glands and is essential

for implantation. The stromal expression of Lif

surrounding the blastocyst at the time of

attachment was also found to be important for

implantation, as described in reference 47.

49. Ernst, M. et al. Defective gp130-mediated signal

transducer and activator of transcription (STAT)

signaling results in degenerative joint disease,

gastrointestinal ulceration, and failure of uterine

implantation. J. Exp. Med. 194, 189–203 (2001).

50. Benson, G. V. et al. Mechanisms of reduced fertility in

Hoxa-10 mutant mice: uterine homeosis and loss of

maternal Hoxa-10 expression. Development 122,

2687–2696 (1996).

51. Hsieh-Li, H. M. et al. Hoxa 11 structure, extensive

antisense transcription, and function in male and

female fertility. Development 121, 1373–1385 (1995).

52. Lim, H., Ma, L., Ma, W. G., Maas, R. L. & Dey, S. K.

Hoxa-10 regulates uterine stromal cell responsiveness

to progesterone during implantation and

decidualization in the mouse. Mol. Endocrinol. 13,

1005–1017 (1999).

53. Satokata, I., Benson, G. & Maas, R. Sexually

dimorphic sterility phenotypes in Hoxa10-deficient

mice. Nature 374, 460–463 (1995).

This paper was the first to show female infertility in

mice that lack Hoxa10. It was later shown that

defective decidualization is the cause of this female

infertility, as described in references 50 and 52.

54. Daikoku, T. et al. Uterine Msx-1 and Wnt4 signaling

becomes aberrant in mice with the loss of leukemia

inhibitory factor or Hoxa-10: evidence for a novel

cytokine-homeobox-Wnt signaling in implantation.

Mol. Endocrinol. 18, 1238–1250 (2004).

This work was the first to provide evidence that

cytokines, homeotic proteins and morphogens in

the mouse uterus constitute a molecular circuitry

that is crucial to implantation.

55. Gendron, R. L. et al. Abnormal uterine stromal and

glandular function associated with maternal

reproductive defects in Hoxa-11 null mice. Biol.

Reprod. 56, 1097–1105 (1997).

56. Taylor, H. S., Arici, A., Olive, D. & Igarashi, P.

HOXA10 is expressed in response to sex steroids at

the time of implantation in the human endometrium.

J. Clin. Invest. 101, 1379–1384 (1998).

57. Wang, W., Van De Water, T. & Lufkin, T. Inner ear and

maternal reproductive defects in mice lacking the

Hmx3 homeobox gene. Development 125, 621–634

(1998).

58. Borthwick, J. M. et al. Determination of the transcript

profile of human endometrium. Mol. Hum. Reprod. 9,

19–33 (2003).

59. Carson, D. D. et al. Changes in gene expression during

the early to mid-luteal (receptive phase) transition in

human endometrium detected by high-density

microarray screening. Mol. Hum. Reprod. 8, 871–879

(2002).

60. Kao, L. C. et al. Global gene profiling in human

endometrium during the window of implantation.

Endocrinology 143, 2119–2138 (2002).

61. Mirkin, S. et al. In search of candidate genes critically

expressed in the human endometrium during the

window of implantation. Hum. Reprod. 20,

2104–2117 (2005).

62. Riesewijk, A. et al. Gene expression profiling of human

endometrial receptivity on days LH+2 versus LH+7

by microarray technology. Mol. Hum. Reprod. 9,

253–264 (2003).

63. Satokata, I. & Maas, R. Msx1 deficient mice exhibit

cleft palate and abnormalities of craniofacial and tooth

development. Nature Genet. 6, 348–356 (1994).

64. Takamoto, N., Zhao, B., Tsai, S. Y. & DeMayo, F. J.

Identification of Indian hedgehog as a progesterone-

responsive gene in the murine uterus. Mol. Endocrinol.

16, 2338–2348 (2002).

65. Matsumoto, H., Zhao, X., Das, S. K., Hogan, B. L. &

Dey, S. K. Indian hedgehog as a progesterone-

responsive factor mediating epithelial-mesenchymal

interactions in the mouse uterus. Dev. Biol. 245,

280–290 (2002).

66. Paria, B. C.

et al. Cellular and molecular responses of

the uterus to embryo implantation can be elicited by

locally applied growth factors. Proc. Natl Acad. Sci.

USA 98, 1047–1052 (2001).

This article provides a comprehensive account of

the expression of morphogens, including HH, BMP,

WNT and FGF signalling in the mouse uterus during

the periimplantation period. The evidence that HH

signalling in the uterine epithelial–mesenchymal

interaction is important for implantation was later

reported in references 64 and 65.

67. Parr, B. A. & McMahon, A. P. Sexually dimorphic

development of the mammalian reproductive tract

requires Wnt-7a. Nature 395, 707–710 (1998).

68. Daikoku, T. et al. Proteomic analysis identifies

immunophilin FK506 binding protein 4 (FKBP52) as a

downstream target of Hoxa10 in the periimplantation

mouse uterus. Mol. Endocrinol. 19, 683–697 (2005).

69. Tranguch, S. et al. Cochaperone immunophilin

FKBP52 is critical to uterine receptivity for embryo

implantation. Proc. Natl Acad. Sci. USA 102,

14326–14331 (2005).

70. Das, S. K. et al. Heparin-binding EGF-like growth

factor gene is induced in the mouse uterus temporally

by the blastocyst solely at the site of its apposition: a

possible ligand for interaction with blastocyst EGF-

receptor in implantation. Development 120,

1071–1083 (1994).

The role of HB-EGF as an early initiator of

molecular crosstalk between the blastocyst and

uterus before attachment was first illustrated in

this study.

71. Iwamoto, R. et al. Heparin-binding EGF-like growth

factor and ErbB signaling is essential for heart function.

Proc. Natl Acad. Sci. USA 100, 3221–3226 (2003).

72. Chobotova, K. et al. Heparin-binding epidermal

growth factor and its receptor ErbB4 mediate

implantation of the human blastocyst. Mech. Dev.

119 , 137–144 (2002).

73. Genbacev, O. D. et al. Trophoblast L-selectin-mediated

adhesion at the maternal-fetal interface. Science 299,

405–408 (2003).

This study shows that, in humans, selectin

oligosaccharide ligands are expressed in the

receptive uterine lining, while the Tr cell surface is

decorated with L-selectin. Further evidence

indicates that this ligand–receptor signalling is

important for human implantation.

74. Fouladi-Nashta, A. A. et al. Characterization of the

uterine phenotype during the peri-implantation period

for LIF-null, MF1 strain mice. Dev. Biol. 281, 1–21

(2005).

75. Lim, H. et al. Multiple female reproductive failures in

cyclooxygenase 2-deficient mice. Cell 91, 197–208

(1997).

This study shows that ovulation, fertilization,

implantation and decidualization are defective in

mice lacking COX2-derived prostaglandins.

76. Lim, H. et al. Cyclo-oxygenase-2-derived prostacyclin

mediates embryo implantation in the mouse via

PPARδ. Genes Dev. 13

, 1561–1574 (1999).

77. Wang, H. et al. Rescue of female infertility from the

loss of cyclooxygenase-2 by compensatory

up-regulation of cyclooxygenase-1 is a function of

genetic makeup. J. Biol. Chem. 279, 10649–10658

(2004).

78. Kim, J. J. et al. Expression of cyclooxygenase-1 and-2

in the baboon endometrium during the menstrual

cycle and pregnancy. Endocrinology 140, 2672–2678

(1999).

79. Song, H. et al. Cytosolic phospholipase A2α is crucial

for ‘‘on-time’’ embryo implantation that directs

subsequent development. Development 129,

2879–2889 (2002).

This work shows that mouse uteri that lack cPLA2α

transiently defer on-time implantation, creating an

adverse ripple effect throughout the course of

pregnancy and leading to poor pregnancy outcome.

A similar phenotype is observed in lpA3-null mice,

as reported in reference 80. The importance of on-

time implantation in human pregnancy outcome is

presented in reference 81.

80. Ye, X. et al. LPA3-mediated lysophosphatidic acid

signalling in embryo implantation and spacing. Nature

435, 104–108 (2005).

81. Wilcox, A. J., Baird, D. D. & Weinberg, C. R. Time of

implantation of the conceptus and loss of pregnancy.

N. Engl. J. Med. 340, 1796–1799 (1999).

82. Hogan, B. L. Bone morphogenetic proteins:

multifunctional regulators of vertebrate development.

Genes Dev. 10, 1580–1594 (1996).

83. Pfendler, K. C., Yoon, J., Taborn, G. U., Kuehn, M. R. &

Iannaccone, P. M. Nodal and bone morphogenetic

protein 5 interact in murine mesoderm formation and

implantation. Genesis 28, 1–14 (2000).

84. Arikawa, T., Omura, K. & Morita, I. Regulation of bone

morphogenetic protein-2 expression by endogenous

prostaglandin E2 in human mesenchymal stem cells. J.

Cell. Physiol. 200, 400–406 (2004).

85. Matsumoto, H. et al. Cyclooxygenase-2 differentially

directs uterine angiogenesis during implantation in

mice. J. Biol. Chem. 277, 29260–29267 (2002).

This study shows that COX2-derived prostaglandins

coordinate VEGF and angiopoietin signalling during

angiogenesis in the mouse deciduum — a process

that is required for the establishment of pregnancy.

86. Paria, B. C. & Dey, S. K. Preimplantation embryo

development in vitro: cooperative interactions among

embryos and role of growth factors. Proc. Natl Acad.

Sci. USA 87, 4756–4760 (1990).

87. Leach, R. E. et al. Pre-eclampsia and expression of

heparin-binding EGF-like growth factor.

Lancet 360,

1215–1219 (2002).

88. National Institutes of Health. Stem Cells: Scientific

Progress and Future Research Directions. Stem Cell

Information [online], http://stemcells.nih.gov/info/

scireport (2001).

89. Gardner, R. L. Specification of embryonic axes begins

before cleavage in normal mouse development.

Development 128, 839–847 (2001).

90. Fujimori, T., Kurotaki, Y., Miyazaki, J. & Nabeshima, Y.

Analysis of cell lineage in two- and four-cell mouse

embryos. Development 130, 5113–5122

(2003).

91. Piotrowska, K., Wianny, F., Pedersen, R. A. &

Zernicka-Goetz, M. Blastomeres arising from the first

cleavage division have distinguishable fates in normal

mouse development. Development 128, 3739–3748

(2001).

92. Piotrowska, K. & Zernicka-Goetz, M. Role for sperm in

spatial patterning of the early mouse embryo.

Nature 409, 517–521 (2001).

93. Gardner, R. L. The early blastocyst is bilaterally

symmetrical and its axis of symmetry is aligned with

the animal–vegetal axis of the zygote in the mouse.

Development 124, 289–301 (1997).

This article proposes the concept of the embryonic

axis and cell polarity during mouse preimplantation

development. The ongoing debate on this subject is

further highlighted in references

89–92,96,101,107 and 108.

94. Piotrowska-Nitsche, K., Perea-Gomez, A., Haraguchi, S.

& Zernicka-Goetz, M. Four-cell stage mouse

blastomeres have different developmental properties.

Development 132, 479–490 (2005).

95. Piotrowska-Nitsche, K. & Zernicka-Goetz, M. Spatial

arrangement of individual 4-cell stage blastomeres

and the order in which they are generated correlate

with blastocyst pattern in the mouse embryo. Mech.

Dev. 122, 487–500 (2005).

96. Plusa, B. et al. The first cleavage of the mouse zygote

predicts the blastocyst axis. Nature 434, 391–395

(2005).

97. Surani, M. A. & Barton, S. C. Spatial distribution of

blastomeres is dependent on cell division order and

interactions in mouse morulae. Dev. Biol. 102,

335–343 (1984).

98. Garbutt, C. L., Johnson, M. H. & George, M. A. When

and how does cell division order influence cell

allocation to the inner cell mass of the mouse

blastocyst? Development 100, 325–332 (1987).

99. Alarcon, V. B. & Marikawa, Y. Unbiased contribution of

the first two blastomeres to mouse blastocyst

development. Mol. Reprod. Dev. 72

, 354–361

(2005).

100. Chroscicka, A., Komorowski, S. & Maleszewski, M.

Both blastomeres of the mouse 2-cell embryo

contribute to the embryonic portion of the blastocyst.

Mol. Reprod. Dev. 68, 308–312 (2004).

101. Motosugi, N., Bauer, T., Polanski, Z., Solter, D. &

Hiiragi, T. Polarity of the mouse embryo is established

at blastocyst and is not prepatterned. Genes Dev. 19,

1081–1092 (2005).

REVIEWS

198

MARCH 2006

VOLUME 7 www.nature.com/reviews/genetics

102. Rossant, J. & Tam, P. P. Emerging asymmetry and

embryonic patterning in early mouse development.

Dev. Cell 7, 155–164 (2004).

103. Zernicka-Goetz, M. First cell fate decisions and spatial

patterning in the early mouse embryo. Semin. Cell

Dev. Biol. 15, 563–572 (2004).

104. Plusa, B., Grabarek, J. B., Piotrowska, K., Glover, D. M.

& Zernicka-Goetz, M. Site of the previous meiotic

division defines cleavage orientation in the mouse

embryo. Nature Cell Biol. 4, 811–815 (2002).

105. Plusa, B., Piotrowska, K. & Zernicka-Goetz, M.

Sperm entry position provides a surface marker for

the first cleavage plane of the mouse zygote. Genesis

32, 193–198 (2002).

106. Davies, T. J. & Gardner, R. L. The plane of first

cleavage is not related to the distribution of sperm

components in the mouse. Hum. Reprod. 17,

2368–2379 (2002).

107. Hiiragi, T. & Solter, D. First cleavage plane of the

mouse egg is not predetermined but defined by the

topology of the two apposing pronuclei. Nature 430,

360–364 (2004).

108. Louvet-Vallee, S., Vinot, S. & Maro, B. Mitotic spindles

and cleavage planes are oriented randomly in the two-

cell mouse embryo. Curr. Biol. 15, 464–469 (2005).

109. Ain, R., Dai, G., Dunmore, J. H., Godwin, A. R. &

Soares, M. J. A prolactin family paralog regulates

reproductive adaptations to a physiological stressor.

Proc. Natl Acad. Sci. USA 101, 16543–16548

(2004).

110. Cheon, Y. P. et al. A genomic approach to identify

novel progesterone receptor regulated pathways in the

uterus during implantation. Mol. Endocrinol. 16,

2853–2871 (2002).

111. Reese, J. et al. Global gene expression analysis to

identify molecular markers of uterine receptivity and

embryo implantation. J. Biol. Chem. 276,

44137–44145 (2001).

112. Reyzer, M. L. & Caprioli, R. M. MALDI mass

spectrometry for direct tissue analysis: a new tool for

biomarker discovery. J. Proteome Res. 4, 1138–1142

(2005).

113 . Ralston, A. & Rossant, J. Genetic regulation of stem

cell origins in the mouse embryo. Clin. Genet. 68,

106–112 (2005).

114. Branford, W. W., Benson, G. V., Ma, L., Maas, R. L. &

Potter, S. S. Characterization of Hoxa-10/Hoxa-11

transheterozygotes reveals functional redundancy and

regulatory interactions. Dev. Biol. 224, 373–387

(2000).

115. Mericskay, M., Kitajewski, J. & Sassoon, D. Wnt5a

is required for proper epithelial–mesenchymal

interactions in the uterus. Development 131,

2061–2072 (2004).

116. Shindo, T. et al. ADAMTS-1: a metalloproteinase-

disintegrin essential for normal growth, fertility, and

organ morphology and function. J. Clin. Invest. 105,

1345–1352 (2000).

117. Simmen, R. C. et al. Subfertility, uterine hypoplasia,

and partial progesterone resistance in mice lacking the

Kruppel-like factor 9/basic transcription element-

binding protein-1 (Bteb1) gene. J. Biol. Chem. 279,

29286–29294 (2004).

118. Fowler, K. J. et al. Uterine dysfunction and genetic

modifiers in centromere protein B-deficient mice.

Genome Res 10, 30–41 (2000).

119. Panda, D. K. et al. Targeted ablation of the

25-hydroxyvitamin D 1α-hydroxylase enzyme:

evidence for skeletal, reproductive, and immune

dysfunction. Proc. Natl. Acad Sci. USA 98,

7498–7503 (2001).

120. Baker, J. et al. Effects of an Igf1 gene null mutation on

mouse reproduction. Mol. Endocrinol. 10, 903–918

(1996).

121. Smith, C. L. et al. Genetic ablation of the steroid

receptor coactivator-ubiquitin ligase, E6-AP, results in

tissue-selective steroid hormone resistance and

defects in reproduction. Mol. Cell Biol. 22, 525–535

(2002).

122. Yoshizawa, T. et al. Mice lacking the vitamin D receptor

exhibit impaired bone formation, uterine hypoplasia

and growth retardation after weaning. Nature Genet.

16, 391–396 (1997).

123. Igakura, T. et al. A null mutation in basigin, an

immunoglobulin superfamily member, indicates its

important roles in peri-implantation development

and spermatogenesis. Dev. Biol. 194, 152–165

(1998).

124. Kuno, N. et al. Female sterility in mice lacking the

basigin gene, which encodes a transmembrane

glycoprotein belonging to the immunoglobulin

superfamily. FEBS Lett. 425, 191–194 (1998).

125. Barak, Y. et al. Effects of peroxisome proliferator-

activated receptor δ on placentation, adiposity, and

colorectal cancer. Proc. Natl Acad. Sci USA 99,

303–308 (2002).

126. Robb, L. et al. Infertility in female mice lacking the

receptor for interleukin 11 is due to a defective uterine

response to implantation. Nature Med 4, 303–308

(1998).

127. Bilinski, P., Roopenian, D. & Gossler, A.

Maternal IL-11Rα function is required for normal

decidua and fetoplacental development in mice.

Genes Dev. 12, 2234–2243 (1998).

128. Meissner, A. & Jaenisch, R. Generation of nuclear

transfer-derived pluripotent ES cells from cloned Cdx2-

deficient blastocysts. Nature 439, 212–215 (2006).

129. Niwa, H. et al. Interaction between Oct3/4 and Cdx2

determines trophectoderm differentiation. Cell 123,

917–929 (2005).

130. Gore, A. V. et al. The zebrafish dorsal axis is apparent at

the four-cell stage. Nature 438, 1030–1035 (2005).

Acknowledgements

We regret that page limitations precluded us from citing

numerous relevant references. The authors’ work embodied

in this article was supported in parts by NIH Grants to S.K.D.

S.K.D. is the recipient of Method to Extend Research in Time

(MERIT) Awards from the National Institute on Drug Abuse

(NIDA) and the National Institute of Child Health and Human

Development (NICHD). H.W. is the recipient of Solvay/Mortola

Research Award from the Society for Gynecologic

Investigation. We thank S. Tranguch for critical reading of the

manuscript.

Competing interests statement

The authors declare no competing financial interests.

DATABASES

The following terms in this article are linked online to:

Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.

fcgi?db=gene

Bmp2 | Cb1 | Cdx2 | Cox1 | Cox2 | cPla2

| Crim1 | Dan | Eomes

| Er

| ErbB1 | ErbB4 | Fgf2 | Ffg10 | Fkbp52 | Flk1 | Hbegf |

Hmx3 | Hoxa10 | Hoxa11 | Ihh | L-selectin | Lif | lpA3 | Msx1 |

Nanog | Noggin | Oct4 | PRA | PRB | sfrp4 | Wnt4 | Wnt7a

UniProtKB: http://ca.expasy.org/sprot/

gp130 | PPARδ | VEGF

FURTHER INFORMATION

Sudhansu K. Dey’s web page: http://www.mc.vanderbilt.

edu/reproductionlab/index.html

NIA Mouse cDNA Project: http://lgsun.grc.nia.nih.gov/

cDNA/CitationFinder.html

SUPPLEMENTARY INFORMATION

See online article: S1 (table)

Access to this links box is available online

REVIEWS

NATURE REVIEWS

GENETICS VOLUME 7

MARCH 2006

199

Transcriptome profiling reveals superovulation with the gonadotropin-releasing hormone agonist trigger impaired embryo implantation in mice

Article

Full-text available

Feb 2024

Introduction Superovulation is a critical step in assisted reproductive technology, but the use of human chorionic gonadotropin (hCG) as a trigger for superovulation can result in ovarian hyperstimulation. Thus, the use of Gonadotropin-releasing hormone agonist (GnRHa) trigger has been increasingly adopted, although it has been associated with a higher rate of pregnancy failure compared to natural cycles. This study aimed to investigate the effect of GnRHa trigger on embryo implantation in a mouse model. Methods Mice in the superovulation (PG) group were administered 7.5 IU of PMSG, followed by the injection of 3.5 μg of GnRHa (Leuprorelin) 48 h later, while mice in the control group (CTR) mated naturally. We compared the number of oocytes, blastocysts, and corpus luteum between the two groups and the implantation sites after the transfer of natural blastocysts. Ovaries, uterus, and serum 2 and 4 days after mating were collected for qRT-PCR, transcriptome sequencing, and hormone assays. Results The PG group had more oocytes, blastocysts, and corpus luteum after superovulation than the CTR group. However, the mRNA expression of leukemia inhibitory factor (Lif) and the number of implantation sites were reduced in the PG group. The ELISA assay revealed that superovulation increased ovarian estrogen secretion. The transcriptome analysis showed that superphysiological estrogen led to a response of the uterus to a high estrogen signal, resulting in abnormal endometrium and extracellular matrix remodeling and up-regulation of ion transport and inflammation-related genes. Conclusion Our findings suggest that a combination of PMSG and GnRHa trigger impaired embryo implantation in mice, as the excessive uterine response to superphysiological estrogen levels can lead to the change of gene expression related to endometrial remodeling, abnormal expression of uterine ion transport genes and excessive immune-related genes.

Stromal Pbrm1 mediates chromatin remodeling necessary for embryo implantation in the mouse uterus

Article

Full-text available

Mar 2024
J CLIN INVEST

Early gestational loss occurs in approximately 20% of all clinically recognized human pregnancies and is an important cause of morbidity. Either embryonic or maternal defects can cause loss, but a functioning and receptive uterine endometrium is crucial for embryo implantation. We report that the switch/sucrose nonfermentable (SWI/SNF) remodeling complex containing polybromo-1 (PBRM1) and Brahma-related gene 1 (BRG1) is essential for implantation of the embryonic blastocyst on the wall of the uterus in mice. Although preimplantation development is unaffected, conditional ablation of Pbrm1 in uterine stromal cells disrupts progesterone pathways and uterine receptivity. Heart and neural crest derivatives expressed 2 (Hand2) encodes a basic helix-loop-helix (bHLH) transcription factor required for embryo implantation. We identify an enhancer of the Hand2 gene in stromal cells that requires PBRM1 for epigenetic histone modifications/coactivator recruitment and looping with the promoter. In Pbrm1cKO mice, perturbation of chromatin assembly at the promoter and enhancer sites compromises Hand2 transcription, adversely affects fibroblast growth factor signaling pathways, prevents normal stromal-epithelial crosstalk, and disrupts embryo implantation. The mutant female mice are infertile and provide insight into potential causes of early pregnancy loss in humans.

Altered DNA methylation and Dnmt expression in obese uterus may cause implantation failure

Article

Full-text available

Jun 2024
J MOL HISTOL

Obesity is defined by increased adipose tissue volume and has become a major risk factor for reproduction. Recent studies have revealed a substantial link between obesity and epigenetics. The epigenome is dynamically regulated mainly by DNA methylation. DNA methylation, which is controlled by DNA methyltransferases (Dnmts), has been widely studied because it is essential for imprinting and regulation of gene expression. In our previous study, we showed that the levels of Dnmt1, Dnmt3a and global DNA methylation was dramatically altered in the testis and ovary of high-fat diet (HFD)-induced obese mice. However, the effect of HFD on Dnmts and global DNA methylation in mouse uterus has not yet been demonstrated. Therefore, in the present study, we aimed to evaluate the effect of HFD on the level of Dnmt1, Dnmt3a, Dnmt3b, Dnmt3l and global DNA methylation in uterus. Our results showed that HFD significantly altered the levels of Dnmts and global DNA methylation in the uterus. The total expression of Dnmt1, Dnmt3a and Dnmt3b was significantly upregulated, while level of Dnmt3l and global DNA methylation were dramatically decreased (p < 0.05). Furthermore, we observed that the expression of Dnmt3b and Dnmt3l was significantly increased in endometrium including gland and epithelium (p < 0.05). Although Dnmt3b was the only protein whose expression significantly increased, the level of global DNA methylation and Dnmt3l significantly decreased in stroma and myometrium (p < 0.05). In conclusion, our results show for the first time that obesity dramatically alters global DNA methylation and expression of Dnmts, and decreased DNA methylation and Dnmt expression may cause abnormal gene expression, especially in the endometrium.

Stromal cell decidualization and embryo implantation: a vulnerable step leading to successful pregnancy

Article

Full-text available

Jun 2024

Jason Zhu

Endometrial stromal cell decidualization is a crucial step in endometrial remodeling during pregnancy. Decidualization is controlled by orchestrated ovarian hormones, followed by the activation of various downstream signaling pathways. Accumulating evidence has shown multiple functions of decidualized endometrial stromal cells during embryo implantation, including tissue remodeling, antioxidative stress, angiogenesis, and immune tolerance. The distinct secretomes of decidualized stromal cells also reveal their intensive interactions with epithelial, endothelial, and immune cells. However, aberrant decidualization leads to pregnancy failures, such as recurrent pregnancy loss and repeated implantation failure. This review aimed to provide an overview of the molecular mechanisms underlying the divergent functions of decidualized endometrial stromal cells and their potential clinical applications. Moreover, the use of single-cell RNA sequencing data further enhances our understanding of these biological processes. This review discusses decidualization-related signaling pathways that serve as potential therapeutic targets for treating implantation failure in in vitro fertilization and provides novel approaches to investigate the underlying causes of female infertility.

Fludrocortisone improves endometrial receptivity by regulating expression of ENaC, SGK1, HAND2, miR-200a, miR-145, miR-451, mTOR, and 4E-BP1 during the implantation window in mice

Article

Apr 2024

Establishment of Murine Pregnancy Requires the Promyelocytic Leukemia Zinc Finger Transcription Factor

Article

Full-text available

Mar 2024
INT J MOL SCI

Using an established human primary cell culture model, we previously demonstrated that the promyelocytic leukemia zinc finger (PLZF) transcription factor is a direct target of the progesterone receptor (PGR) and is essential for progestin-dependent decidualization of human endometrial stromal cells (HESCs). These in vitro findings were supported by immunohistochemical analysis of human endometrial tissue biopsies, which showed that the strongest immunoreactivity for endometrial PLZF is detected during the progesterone (P4)-dominant secretory phase of the menstrual cycle. While these human studies provided critical clinical support for the important role of PLZF in P4-dependent HESC decidualization, functional validation in vivo was not possible due to the absence of suitable animal models. To address this deficiency, we recently generated a conditional knockout mouse model in which PLZF is ablated in PGR-positive cells of the mouse (Plzf d/d). The Plzf d/d female was phenotypically analyzed using immunoblotting, real-time PCR, and immunohistochemistry. Reproductive function was tested using the timed natural pregnancy model as well as the artificial decidual response assay. Even though ovarian activity is not affected, female Plzf d/d mice exhibit an infertility phenotype due to an inability of the embryo to implant into the Plzf d/d endometrium. Initial cellular and molecular phenotyping investigations reveal that the Plzf d/d endometrium is unable to develop a transient receptive state, which is reflected at the molecular level by a blunted response to P4 exposure with a concomitant unopposed response to 17-β estradiol. In addition to a defect in P4-dependent receptivity, the Plzf d/d endometrium fails to undergo decidualization in response to an artificial decidual stimulus, providing the in vivo validation for our earlier HESC culture findings. Collectively, our new Plzf d/d mouse model underscores the physiological importance of the PLZF transcription factor not only in endometrial stromal cell decidualization but also uterine receptivity, two uterine cellular processes that are indispensable for the establishment of pregnancy.

H3K27me3 timely dictates uterine epithelial transcriptome remodeling and thus transformation essential for normal embryo implantation

Article

Full-text available

May 2024
CELL DEATH DIFFER

A GREB1-steroid receptor feedforward mechanism governs differential GREB1 action in endometrial function and endometriosis

Article

Full-text available

Mar 2024

Cellular responses to the steroid hormones, estrogen (E2), and progesterone (P4) are governed by their cognate receptor’s transcriptional output. However, the feed-forward mechanisms that shape cell-type-specific transcriptional fulcrums for steroid receptors are unidentified. Herein, we found that a common feed-forward mechanism between GREB1 and steroid receptors regulates the differential effect of GREB1 on steroid hormones in a physiological or pathological context. In physiological (receptive) endometrium, GREB1 controls P4-responses in uterine stroma, affecting endometrial receptivity and decidualization, while not affecting E2-mediated epithelial proliferation. Of mechanism, progesterone-induced GREB1 physically interacts with the progesterone receptor, acting as a cofactor in a positive feedback mechanism to regulate P4-responsive genes. Conversely, in endometrial pathology (endometriosis), E2-induced GREB1 modulates E2-dependent gene expression to promote the growth of endometriotic lesions in mice. This differential action of GREB1 exerted by a common feed-forward mechanism with steroid receptors advances our understanding of mechanisms that underlie cell- and tissue-specific steroid hormone actions.

The regulated cell death at the maternal-fetal interface: beneficial or detrimental?

Article

Full-text available

Feb 2024

Regulated cell death (RCD) plays a fundamental role in placental development and tissue homeostasis. Placental development relies upon effective implantation and invasion of the maternal decidua by the trophoblast and an immune tolerant environment maintained by various cells at the maternal-fetal interface. Although cell death in the placenta can affect fetal development and even cause pregnancy-related diseases, accumulating evidence has revealed that several regulated cell death were found at the maternal-fetal interface under physiological or pathological conditions, the exact types of cell death and the precise molecular mechanisms remain elusive. In this review, we summarized the apoptosis, necroptosis and autophagy play both promoting and inhibiting roles in the differentiation, invasion of trophoblast, remodeling of the uterine spiral artery and decidualization, whereas ferroptosis and pyroptosis have adverse effects. RCD serves as a mode of communication between different cells to better maintain the maternal-fetal interface microenvironment. Maintaining the balance of RCD at the maternal-fetal interface is of utmost importance for the development of the placenta, establishment of an immune microenvironment, and prevention of pregnancy disorders. In addition, we also revealed an association between abnormal expression of key molecules in different types of RCD and pregnancy-related diseases, which may yield significant insights into the pathogenesis and treatment of pregnancy-related complications.

Spatial and molecular anatomy of the endometrium during embryo implantation: a current overview of key regulators of blastocyst invasion

Article

Feb 2024
FEBS J

Embryo implantation is composed of three steps: blastocyst apposition, adhesion/attachment and invasion. Blastocyst invasion has been studied less extensively than the other two events. Historically, studies conducted using electron microscopy have shown the removal of epithelial cells in the vicinity of the attached blastocysts in rodents, although the underlying mechanisms have remained unclear. Here, we describe recent studies using mice with uterine‐specific gene deletion that demonstrated important roles for nuclear proteins such as progesterone receptor, hypoxia inducible factor and retinoblastoma in the regulation of embryo invasion. In these mouse models, the detachment of the endometrial luminal epithelium, decidualization in the stroma, and the activation of trophoblasts have been found to be important in ensuring embryo invasion. This review summarizes the molecular signaling associated with these cellular events, mainly evidenced by mouse models.

Expression of Cyclooxygenase1 and -2 in the Baboon Endometrium during the Menstrual Cycle and Pregnancy

Article

Full-text available

Jun 1999
ENDOCRINOLOGY

Cyclooxygenase (COX) is the rate-limiting enzyme in the biosyn- thesis of PGs. PGs together with ovarian steroids play important regulatory roles in the establishment and maintenance of pregnancy in a number of different species. In the primate, little is known about the role of PGs in these processes. In this study, the uterine expression of COX-1 and COX-2 throughout the menstrual cycle (late follicular, day 5 postovulation (PO), day 10 PO, and day 14 PO) and pregnancy (days 12-18, day 39, day 51, and near term) was analyzed using semiquantitative RT-PCR, in situ hybridization, and immunocyto- chemistry. During the menstrual cycle, the highest expression of COX-1 occurred in luteal phase endometrium and was localized to the surface and glandular epithelium. The stromal cells did not express detectable levels of COX-1 at any time. COX-2 messenger RNA (mRNA) expression, as measured by RT-PCR, was evident at all stages of the menstrual cycle, and in situ hybridization showed spe- cific localization for this mRNA in the epithelial cells during the cycle. Treatment of animals with the antiprogestin (ZK 137.316) for 9 days (beginning on the day of the LH surge) inhibited COX-1 expression in the epithelium when the tissue was analyzed on day 10 PO, whereas COX-2 expression disappeared in the epithelium and increased in the stroma. With the onset of pregnancy, COX-1 expression in epithelial cells decreased dramatically. In contrast, COX-2 continued to be de- tected on the surface epithelium and was also strongly expressed specifically in the stromal cells at the site of implantation. Immuno- cytochemical staining for COX-2 showed the same pattern of expres- sion for the protein as the message. Finally, near-term decidua ex- pressed very little COX-1 or COX-2 mRNA. These studies suggest that in the baboon endometrium, COX-1 expression is regulated primarily by progesterone, whereas regulation of COX-2 expression may involve additional mediators of embryonic origin at the site of implantation. (Endocrinology 140: 2672-2678, 1999)

Uterine Decidual Response Occurs in Estrogen Receptor Deficient Mice

Article

Full-text available

Jun 1999
ENDOCRINOLOGY

Embryo-uterine interactions leading to the attachment reaction is followed by stromal cell proliferation and differentiation into decidual cells (decidualization) at the sites of blastocyst apposition. In rodents, decidualization is also induced by application of an artificial stimulus (intraluminal oil infusion) in a pseudopregnant uterus, or to one that has been appropriately prepared by exogenous progesterone (P4) and estrogen. The process of decidualization is under the control of these steroids in the presence of blastocysts or deciduogenic stimuli. Al- though it is well known that estrogen is required for the induction of progesterone receptors in the uterus, the functional importance of estrogen in the process of decidualization is poorly understood. To better understand the role of estrogenic actions in decidualization, we used wild-type and estrogen receptor-a knock-out (ERKO) mice for induction of decidualization employing a defined steroid hormonal treatment schedule. Our results demonstrate that P4 alone induces decidualization in ovariectomized wild-type or ERKO mice in re- sponse to intraluminal oil infusion in the absence of estrogen. A combined treatment of either estradiol-17b (E2) or its catecholme- tabolite 4-hydroxyestradiol-17b (4-OH-E2) with P4 does not potentiate the decidual response produced by P4 treatment alone in either ovari- ectomized wild-type or ERKO mice. The induction of decidual re- sponse was associated with up-regulation of decidual cell marker genes, such as progesterone receptor, metallothionein-1, and cyclo- oxygenase-2. The results suggest that the stromal cell sensitivity to decidualization is critically dependent on P4-regulated events, and estrogenic induction of progesterone receptor via classical nuclear ER-a is not critical for this process. (Endocrinology 140:2704 -2710, 1999)

Eomesodermin is required for mouse trophoblast development and mesoderm formation

Article

Full-text available

Mar 2000

The earliest cell fate decision in the mammalian embryo separates the extra-embryonic trophoblast lineage, which forms the fetal portion of the placenta, from the embryonic cell lineages. The body plan of the embryo proper is established only later at gastrulation, when the pluripotent epiblast gives rise to the germ layers ectoderm, mesoderm and endoderm. Here we show that the T-box gene Eomesodermin performs essential functions in both trophoblast development and gastrulation. Mouse embryos lacking Eomesodermin arrest at the blastocyst stage. Mutant trophoectoderm does not differentiate into trophoblast, indicating that Eomesodermin may be required for the development of trophoblast stem cells. In the embryo proper, Eomesodermin is essential for mesoderm formation. Although the specification of the anterior- posterior axis and the initial response to mesoderm-inducing signals is intact in mutant epiblasts, the prospective mesodermal cells are not recruited into the primitive streak. Our results indicate that Eomesodermin defines a conserved molecular pathway controlling the morphogenetic movements of germ layer formation and has acquired a new function in mammals in the differentiation of trophoblast.

Corrigendum: The zebrafish dorsal axis is apparent at the four-cell stage

Article

Dec 2012

Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening

Article

Sep 2002
MOL HUM REPROD

D. D. Carson

High density cDNA microarray screening was used to determine changes in gene expression occurring during the transition between the early luteal (prereceptive) and mid-luteal (receptive) phases in human endometrium. Of ~12 000 genes profiled, 693 (5.8%) displayed >2-fold differences in relative levels of expression between these stages. Of these, 370 genes (3.1%) displayed decreases ranging from 2- to >100-fold while 323 genes (2.7%) displayed increases ranging from 2- to >45-fold. Many genes correspond to mRNAs encoding proteins previously shown to change in a similar manner between the proliferative and mid-luteal phases, serving as one validation of the microarray screening results. In addition, novel genes were identified. Genes encoding cell surface receptors, adhesion and extracellular matrix proteins and growth factors accounted for 20% of the changes. Several genes were studied further by Northern blot analyses. These results confirmed that claudin-4/Clostridium perfringens enterotoxin (CPE) receptor and osteopontin (OPN) mRNA increased ~4- and 12-fold respectively, while betaig-H3 (BIGH3) decreased >80% during the early to mid-luteal transition. Immunostaining also revealed strong specific staining for claudin-4/CPE, EP1 and prostaglandin receptor in epithelia, and leukotriene B4 receptor in both epithelia and stroma, at the mid-luteal stage. Collectively, these studies identify multiple new candidate markers that may be used to predict the receptive phase in humans. Some of these gene products, e.g. OPN, may play direct roles in embryo–uterine interactions during the implantation process.

Dysregulation of EGF Family of Growth Factors and COX-2 in the Uterus during the Preattachment and Attachment Reactions of the Blastocyst with the Luminal Epithelium Correlates with Implantation Failure in LIF- Deficient Mice

Article

Aug 2000

Haengseok Song

Global Gene Profiling in Human Endometrium during the Window of Implantation

Article

Jun 2002

L. C. Kao

Coordination of Differential Effects of Primary Estrogen and Catecholestrogen on Two Distinct Targets Mediates Embryo Implantation in the Mouse

Article

Dec 1998

B. C. Paria

In the mouse, estrogen is essential for blastocyst implantation in the progesterone (P4)-primed uterus. The mechanism(s) by which estrogen initiates this response still remains elusive. The present investigation, using delayed implantation in the mouse, examined the differential role of estradiol-17β (E2) and its catechol metabolite 4-hydroxy-E2 (4-OH-E2) in uterine and blastocyst activation for implantation. The conditions of delayed implantation were induced by ovariectomizing mice on day 4 (day 1 = vaginal plug) of pregnancy or pseudopregnancy and maintaining them with P4 from days 5–7. The binding of EGF to blastocysts was used as a marker for blastocyst activation. Our results show that whereas E2 fails to activate dormant blastocysts (with respect to EGF binding in vitro), 4-OH-E2, cAMP, or prostaglandin E2, is effective in this response. Further, whereas 4-OH-E2 induced-activation is not blocked by an antiestrogen, an inhibitor of PG synthesis, adenylyl cyclase or protein kinase A effectively blocks this activation. These results suggest that 4-OH-E2 effects on blastocysts are mediated by PGs, which, in turn, stimulate cAMP production and thus activation of protein kinase A. Two-fluoro-E2 is a poor substrate and an inhibitor of catecholestrogen synthesis, but it is estrogenic, with respect to uterine growth and gene expression. Using blastocyst transfer experiments, we observed that dormant blastocysts incubated with 4-OH-E2 in vitro, but not with E2, are capable of implanting in P4-treated delayed implanting mice receiving two-fluoro-E2. The results suggest that whereas E2 is necessary for preparation of the uterus, uterine-derived catecholestrogen is important for blastocyst activation for implantation. Indeed, the receptive uterus has the capacity to synthesize 4-OH-E2. Collectively, we demonstrate that the primary ovarian estrogen E2, via its interaction with nuclear estrogen receptors, participates in the preparation of the P4-primed uterus to the receptive state in an endocrine manner, whereas its metabolite 4-OH-E2, produced from E2 in the uterus, mediates blastocyst activation for implantation in a paracrine manner. Our results also establish that these target-specific effects of primary estrogen and catecholestrogen are both essential for implantation and that successful implantation occurs only when the activated stage of the blastocyst coincides with the receptive state of the uterus.

Determination of the transcript profile of human endometrium

Article

Jan 2003
MOL HUM REPROD

Jane M. Borthwick

The response of the human endometrium to the ovarian hormones, estrogen and progesterone, has been the focus of decades of research. In order to understand this critical aspect of endometrial physiology, we undertook a genome-wide analysis of transcript abundance and changes in transcript level between normal endometrium in the proliferative and secretory phases of the menstrual cycle. A high-density, oligonucleotide gene array, comprising 60 000 gene targets, was used to define the gene expression profile of proliferative and secretory phase endometrium. Results from the arrays were verified using real-time PCR. The expression levels of 149 transcripts differed significantly between the two phases of the cycle determined by stringent range limits (99.99%), calculated using local variance values. These transcripts include previously documented steroidally responsive genes (such as placental protein 14 and stromelysin-3) and novel transcripts not previously linked to either endometrial physiology or steroid regulation (such as intestinal trefoil factor and a number of expressed sequence tags). Examination of the 5¢ promoter regions of these genes identified many putative estrogen and progesterone receptor DNA binding domains, suggesting a direct response of these genes to the ovarian hormones.

Multiple Female Reproductive Failures in Cyclooxygenase 2–Deficient Mice

Article

Oct 1997
CELL

Cyclooxygenase (COX) is the rate-limiting enzyme in the synthesis of prostaglandins (PGs) and exists in two isoforms, COX-1 and COX-2. In spite of long-standing speculation, definitive roles of PGs in various events of early pregnancy remain elusive. We demonstrate herein that the targeted disruption of COX-2, but not COX-1, in mice produces multiple failures in female reproductive processes that include ovulation, fertilization, implantation, and decidualization. Using multiple approaches, we conclude that these defects are the direct result of target organ–specific COX-2 deficiency but are not the result of deficiency of pituitary gonadotropins or ovarian steroid hormones, or reduced responsiveness of the target organs to their respective hormones.

Wang H, Dey SK. Roadmap to embryo implantation: clues from mouse models. Nat Rev Genet 7: 185-199

Abstract

Recommended publications

Molecular and cellular aspects of blastocyst dormancy and reactivation for implantation

Embryo–Uterine Interactions during Implantation: Potential Sites of Interference by Environmental To...

Cell and molecular regulation of the mouse blastocyst

Trophoblast and hypoblast in the monotreme, marsupial and eutherian mammal: Evolution and origins

Role of CDX2 and cell polarity in cell allocation and specification of trophectoderm and inner cell...