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Understanding implantation window, a crucial
phenomenon
Alok Sharma,
Pratap Kumar1
Department of Obstetrics
and Gynaecology, Deen
Dayal Upadhyaya Hospital,
Shimla, Himachal Pradesh,
1
Department of Obstetrics
and Gynaecology, Kasturba
Medical College, Manipal
University, Manipal,
Karnataka, India
Address for correspondence:
Dr. Pratap Kumar,
Professor, Department of
Obstetrics and Gynecology,
Kasturba Medical College,
Manipal University, Manipal
576104, Karnataka, India.
E-mail: pratap.kumar@
manipal.edu
Received: 13.03.12
Review completed: 13.03.12
Accepted: 15.03.12
Access this article online
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Website:
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DOI:
10.4103/0974-1208.97777
2 Journal of Human Reproductive Sciences / Volume 5 / Issue 1 / Jan - Apr 2012
Review Article
ABSTRACT
Embryo implantation is a well-defined and precise process, in which various factors
come into play one after the other. There is only a specific period of time during which
implantation is possible i.e. “implantation window”. Selectins were proposed to have an
important role in this phase to ensure suitable rolling of the blastocyst. To prevent the
blastocyst from adhering to an area with poor chances of implantation, an important
role is played by the repellent activity of MUC-1, which is widely expressed throughout
the endometrium and, surprisingly, even increases before implantation. In particular
endometrial areas, secretion of chemokines and growth factors will attract the blastocyst
to landing platforms known as pinopods. These pinopods are fully developed for only
1 or 2 days and extend over the tips of the microvilli expressing the repellent MUC-1.
At this stage, adhesion molecules such as integrins and cadherins intervene to ensure
adhesiveness between the embryo and the endometrium.
KEY WORDS: Cellular adhesion molecule family, decidua, implantation
INTRODUCTION structurally and in the composition of its
secretions.
Embryo implantation represents the most
critical step of the reproductive process Adaptation of uterus
in many species. It consists of a unique To enable implantation, the uterus goes
biological phenomenon, by which the through changes in order to be able to receive
blastocyst becomes intimately connected to the embryo.
the maternal endometrial surface to form
the placenta that will provide an interface Predecidualization
between the growing fetus and the maternal The endometrium increases thickness,
circulation. [1] Successful implantation becomes more vascularized and its glands
requires a receptive endometrium, a normal grow to be tortuous and boosted in their
and functional embryo at the blastocyst secretions. These changes reach their
developmental stage and a synchronized maximum about 7 days after ovulation.
dialogue between maternal and embryonic
tissues. Implantation occurs about 9 days Decidualization
after ovulation, ranging between 6 to Decidualization succeeds predecidualiza-
12 days.[2] tion if pregnancy occurs. This is an
expansion of it, further developing the
The implantation window uterine glands, the zona compacta and the
There are many conditions that must epithelium of decidual cells lining it. The
be satisfied in order for a successful decidual cells become filled with lipids and
implantation to take place. There is only glycogen and take the polyhedral shape
a specific period of time during which characteristic for decidual cells. It is likely
implantation is possible; [3] this is the that the blastocyst itself makes the main
“implantation window”. The implantation contribution to this additional growing and
window is started by preparations in sustaining of the decidua. An indication
the endometrium of the uterus, both of this is that decidualization occurs at a
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Sharma and Kumar: Complexity of implantation window
higher degree in conception cycles than in non-conception
cycles. Furthermore, similar changes are observed when
giving stimuli mimicking the natural invasion of the
embryo.[3]
Decidua throughout pregnancy
After implantation, the decidua remains at least through the
first trimester. However, its most prominent time is during
the early stages of pregnancy, during implantation. Its
function as a surrounding tissue is replaced by the definitive
placenta. However, some elements of the decidualization
remain throughout pregnancy.[3]
Pinopodes (Uterodomes)
Pinopods are bleb-like protrusions found on the apical
surface of the endometrial epithelium.[4] They appear
between day 19 and day 21 of gestational age.[3] This
corresponds to a fertilization age of approximately
5 to 7 days, which corresponds well with the time of
implantation. They only persist for 2 to 3 days.[3] The
development of them is enhanced by progesterone. These
structures are several micrometers wide and project into
the uterine lumen above the microvilli level. They were first
described in mice[5] and later in human endometrium.[6,7]
The term ‘pinopod’, derived from the Greek word ‘drinking
foot’. Electron microscopy is the major tool used to observe
these structures.[6,7] Pinopod expression is limited to a
brief period of maximum 2 days in the menstrual cycle
corresponding to the putative window of implantation.[8,9]
Others have detected that pinopods are present throughout
the mid- to late-secretory phase, however, displaying cycle-
dependent morphological changes. This suggests that
morphology, rather than pinopod presence or absence, is
of great significance.[4] The pinopod-regulated expression
pattern throughout the menstrual cycle advocates their use
as markers of implantation.
The detection of pinopods during the mid-secretory
phase may be extremely useful for an assessment of
endometrial receptivity to optimize implantation rates.
Pinopods endocytose uterine fluid and macromolecules
in it. By doing so, the volume of the uterus decreases,
taking the walls closer to the embryoblast floating in it.
Thus, the period of active pinocytes might also limit the
implantation window. [3] Pinopods continue to absorb
fluid, and removes most of it during the early stages of
implantation.
Nourishment
The embryoblast spends approximately 72 hours in the
uterine cavity before implanting. In that time, it cannot
receive nourishment directly from the blood of the mother,
and must rely on secreted nutrients into the uterine cavity,
e.g. iron and fat-soluble vitamins.[3]
Journal of Human Reproductive Sciences / Volume 5 / Issue 1 / Jan - Apr 2012
Growth and implantation
In addition to nourishment, the endometrium secretes
several steroid-dependent proteins, important for growth
and implantation. Cholesterol and steroids [3] are also
secreted. Implantation is further facilitated by synthesis
of matrix substances, adhesion molecules and surface
receptors for the matrix substances.
Mechanism
Implantation is initiated when the blastocyst comes into
contact with the uterine wall. The process of implantation
may be classified into 3 stages: Apposition, adhesion and
invasion.[10] During blastocyst apposition, trophoblast
cells adhere to the receptive endometrial epithelium. The
blastocyst will subsequently anchor to the endometrial basal
lamina and stromal extracellular matrix (ECM). At this point,
the achieved embryo–endometrial linkage can no longer be
dislocated by uterine flushing. This is followed by an invasive
blastocyst penetration through the luminal epithelium.[10]
Even though the blastocyst can implant in different human
tissues, surprisingly in the endometrium, this phenomenon
can only occur during a self-limited period spanning
between days 20 and 24 of a regular menstrual cycle (day
LH + 7 to LH + 11). Throughout this period, namely the
window of implantation,[11] the human endometrium is
primed for blastocyst attachment, given that it has acquired
an accurate morphological and functional state initiated by
ovarian steroid hormones.[12]
Zona hatching
To be able to perform implantation, the blastocyst first needs
to get rid of its zona pellucida. This process can be called
“hatching”. A substance probably involved is plasmin.
Plasminogen, the plasmin precursor, is found in the uterine
cavity, and blastocyst factors contribute to its conversion to
active plasmin. This hypothesis is supported by lytic effects
in vitro by plasmin.[3]
Apposition
The very first, albeit loose, connection between the
blastocyst and the endometrium is called the apposition.[3]
Location On the endometrium, the apposition is usually
made where there is a small crypt in it, perhaps because
it increases the area of contact with the rather spherical
blastocyst. On the blastocyst, on the other hand, it occurs
at a location where there has been enough lysis of the zona
pellucida to have created a rupture to enable direct contact
between the underlying trophoblast and the decidua of the
endometrium.[3]
Adhesion
Adhesion is a much stronger attachment to the endometrium
3
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Sharma and Kumar: Complexity of implantation window
than the loose apposition. The trophoblasts adhere
by penetrating the endometrium, with protrusions of
trophoblast cells.
Cellular adhesion molecules family
The cellular adhesion molecule (CAM) family is composed
of 4 members known as integrins, cadherins, selectins,
and immunoglobulins. These surface ligands, usually
glycoproteins, mediate cell-to-cell adhesion. Their classical
functions include maintenance of tissue integration, wound
healing, morphogenic movements, cellular migrations, and
tumor metastasis.
Integrins
Integrins are a family of transmembrane glycoproteins.
A large variety of integrins have been described within the
luminal and glandular endometrial epithelium.[13] Whereas
the majority of the integrins are constitutively expressed
throughout the entire menstrual cycle, others exhibit an
interesting regulated pattern within the cycle.[13] Integrins
whose expression is increased in the mid-luteal phase
were proposed as markers for the frame of the window of
implantation.[14] 3 cycle-specific integrins are co-expressed
by the human endometrium defined histologically on days
20 – 24 of the human menstrual cycle: a1b1, a4b1 and aVb3.
In regard to its expression pattern along with its epithelial
localization, aVb3 has been proposed as a potential receptor
for embryonic attachment.[15]
Integrins are also expressed by the human trophoblast
at the time of implantation.[16] Considering expression
and regulation, aVb3 represents a promising clinical and
research marker of the human implantation process.
Selectins
Selectins are glycoproteins, which also belong to the CAM
family. They include P-selectin, L-selectin, and E-selectin.
The human L-selectin is of importance in the implantation
process.[17]
Cadherins
Cadherins constitute a group of glycoproteins responsible
for the calcium-dependent cell-to-cell adhesion mechanism.
They are divided into subclasses E-, P-, and N-cadherins
that are distinct in immunological specificity and in
tissue distribution. In regard to implantation, E-cadherin
represents the most studied subclass.
Immunoglobulins
Among the CAMs family, the immunoglobulins superfamily
is the most extensive. Intercellular adhesion molecule-1
(ICAM-1 or CD54) is a transmembrane glycoprotein
that belongs to the immunoglobulin superfamily and is
constitutively expressed on the cell surface of a variety of
4 Journal of Human Reproductive Sciences / Volume 5 / Issue 1 / Jan - Apr 2012
cell types, such as fibroblasts, leukocytes, endothelial, and
epithelial cells.[18]
Although ICAM-1 was not shown to be indispensable for the
early steps of blastocyst interactions with the endometrium,
it could participate indirectly in this process by interacting
with the immune system. A clearer picture of human
endometrial pathophysiologies may be acquired by further
studies of ICAM-1 expression and function.
Mucins
Mucins are high molecular weight (MW) glycoproteins.
Among the 14 cloned human mucins, only Mucin-1 (MUC1)
and to a lesser extent, MUC6 have been found in the human
endometrium.[19] MUC1 appears to be a negative factor
for an embryo implantation. Indeed, in the area where
implantation takes place, MUC1 disappears. This effect
was shown to be controlled in vitro mainly by the sheddase
family enzymes that are modulated by blastocyst and
endometrial derived factors. Because endometrial MUC1
increases at the time of implantation, it has been suspected
that this factor has a crucial role to direct the embryo
temporally and spatially to effective implantation.
Cytokines (TH1, TH2, Tregs)
Cytokines comprise a group of proteins that separately
or in concert modulate a variety of cellular functions,
such as cellular proliferation and differentiation. They
play a major role in the reparative and inflammatory-like
processes occurring every menstrual cycle in the human
endometrium, but they are also implicated in critical
reproductive events such as ovulation and implantation.[20]
T cells have also been a focus of research. Initially, T cells
were thought to influence implantation through a TH1/TH2
balance.[21] Pregnancy was postulated to be a TH2-mediated
event; TH1 cytokines such IFN-γ and TNF-a are associated
with infertility and abortion, and these effects can be
reversed in mice by injecting the TH2 cytokine IL-10. [22]
Tregs may play a role in implantation and are essential for
the establishment of peripheral tolerance; they suppress
(auto-) reactive T cells.[23] Tregs have been characterized as
CD4+CD25+ cells.[24] Forkhead box p3 (Foxp3) is present
exclusively in Tregs and is necessary for their development
and function.[25] In the context of pregnancy, Tregs have
been characterized as essential to the establishment of
allotolerance. CD4+CD25+ T cells have been found in the
human decidua at various stages of pregnancy. Their levels
are highest in the peripheral blood during the first trimester
of pregnancy. [26] Furthermore, autoimmune diseases
improve during the course of pregnancy and subsequently
relapse after delivery.[27] In a recent experiment, endometrial
biopsies of (unexplained) infertile and proven fertile women
are compared for the expression of transcription factors that
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Sharma and Kumar: Complexity of implantation window
determine T cell differentiation. It was found that expression
of Foxp3, necessary for Treg development and function, was
significantly lower in the infertile group of women, whereas
the transcription factors T-bet and GATA-3 (for TH1 and
TH2, respectively) did not differ between both groups.[28]
This supports the pivotal role of Tregs in successful human
implantation. In conclusion, Tregs are of key importance
to successful establishment of pregnancy. Much research
is still needed to identify the precise roles of CD4+CD25+
T cells and possibly other subsets of Tregs.
Leukemia inhibiting factor
Leukemia inhibiting factor (LIF) is an IL-6 family pleiotropic
cytokine, which also includes oncostatin M (OSM), ciliary
neurotrophic factor (CNTF), and cardiotrophin 1. A
recombinant human LIF (r-hLIF) has been investigated
in preclinical and clinical trials to improve endometrial
receptivity in RIF patients. In view of the important role of
LIF in implantation, an administration of such r-hLIF could
be valuable in future studies.
Interleukins
IL-6 is a pleiotropic cytokine, originally identified as a factor
inducing immunoglobulin production in activated B cells and
initially designated as IFN-b2 and B-cell differentiation factor
or B-cell stimulatory factor-2. The fact that IL-6 is maximally
expressed[29] during the window of implantation. IL-1 system
may be an important paracrine/autocrine mediator of local
intercellular interactions in the endometrial tissue.[30]
Colony stimulating factor-1
Colony stimulating factor-1 (CSF-1) expression and receptors
for CSF-1 are found both in human endometrium (peaking
in decidua) and in the pre-implantation embryo. Mice with
aninactivating mutation in the CSF-1 gene are infertile
because of low rates of implantation and fetal viability.[31]
Prostaglandins
The process of implantation can be thought of as a
proinflammatory reaction,[32] given that embryo attachment
and invasion into the endometrium require connection to the
maternal vascular system. It has long been speculated that
prostaglandins (PGs), as vasoactive factors, play an important
role in ovulation, fertilization, and in late-pregnancy
processes leading to the onset of labor.[33] PGs are members
of the ‘eicosanoids’ family, which also comprises leukotrienes
(LTs) and thromboxanes (TXa). They consist of 4 members,
named PGD2, PGE2, PGF2a and prostacyclin (PGI2), which
are generated from the membrane phospholipids by the
consecutive action of two enzymes, cytosolic phospholipase
A2(cPLA2) and cyclooxygenase (COX).
The lack of either of these enzymes leads to an absence of PG
synthesis, which then results in several implantation defects.
Journal of Human Reproductive Sciences / Volume 5 / Issue 1 / Jan - Apr 2012
PGs were shown to be essential for embryo implantation.
Their role consists in timing the window of implantation.
Delayed timing of blastocyst implantation has a ripple
effect that presents in mice as embryo crowding near the
cervix, abnormal placentation, and fetal resorption. PGs
supplementation can partially restore a normal phenotype.
Whether PGs have a similar role in human implantation
should be further explored.
Proteoglycan receptors
another ligand-receptor system involved in adhesion is
proteoglycan receptors, found on the surface of the decidua
of the uterus. Their counterparts, the proteoglycans, are
found around the trophoblast cells of the blastocyst.
This ligand-receptor system also is present just at the
implantation window.[3]
Invasion
Invasion is an even further establishment of the blastocyst
in the endometrium.
Syncytiotrophoblasts
The protrusions of trophoblast cells that adhere into the
endometrium continue to proliferate and penetrate into
the endometrium. These penetrating cells differentiate
to become a new type of cells, syncytiotrophoblast. The
prefix syn- refers to that the boundaries between these
cells disappears, forming a single mass of a multitude
of cell nuclei (a syncytium). The rest of the trophoblasts,
surrounding the inner cell mass, are hereafter called
cytotrophoblasts.
Invasion continues with the syncytiotrophoblasts reaching
the basal membrane beneath the decidual cells, penetrating
it, and further invading into the uterine stroma. Finally, the
whole embryo is embedded in the endometrium. Eventually,
the syncytiotrophoblasts come into contact with maternal
blood and form chorionic villi. This is the initiation of
forming the placenta.
CONCLUSION
Embryo implantation is the result of a well-orchestrated
sequence of events including cellular adhesion, invasion, and
immune regulatory mechanisms, some of which are controlled
through genetic processes by the ovarian hormones. It is
proposed that an embryo implantation is a well-defined and
precise process, in which various factors come into play one
after the other, yet remaining in close collaboration.
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How to cite this article: Sharma A, Kumar P. Understanding implantation
window, a crucial phenomenon. J Hum Reprod Sci 2012;5:2-6.
Source of Support: Nil, Conflict of Interest: None declared.