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Human implantation
 Implantation of the human embryo

Alex Lopata
Department of Obstetrics and Gynaecology, University of Melbourne,
Royal Women's Hospital, 132 Grattan Street, Carlton, Victoria 3053,

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Australia

Embryonic development and implantation

For information on the growth of the early human embryo in the genital tract
we still depend on the specimens collected by Hertig et al. (1956). In these
classical studies embryos were obtained from the uterus or Fallopian tubes, after
hysterectomies performed at estimated times following ovulation and conception
times based on recorded coital dates. On about day 3 after ovulation a 12-cell
embryo, possibly 60-72 h of age and described as a morula, was recovered from
the uterus. By day 4 after presumed ovulation an early blastocyst, containing 53
trophoblastic and five embryonic cells was obtained. While by day 4.5 .after
inferred ovulation, a zona-free blastocyst, containing 99 trophoblastic and eight
embryonic cells, was recovered from the uterus (Hertig et al., 1956). In these
and earlier studies the time of ovulation was estimated from menstrual cycle
data as well as endometrial and corpus luteum morphology (Hertig et al., 1954).
More recently, collecting embryos and ova from the uterus by non-surgical
procedures, after more precise detection of the expected times of ovulation, was
reported by two groups (Croxatto et al., 1972; Buster et ai., 1985). Croxatto
et al. used urinary luteinizing hormone (LH) and pregnanediol assays, and other
parameters, to predict ovulation which, in many cases, was also assumed to
correspond to the time of fertilization. In one woman they found a l2-16-cell
morula 4 days after the LH peak and 3 days after coitus, and in a second case a
16-cell morula which was considered to be 4-5 days old. They also recovered
an expanded zonal blastocyst, containing at least 186 cells, which was considered
to be 5 days or slightly older. Buster et al. (1985) used serum LH assays and
artificial inseminations to estimate ovulation and fertilization times. Embryos
were collected by non-surgical uterine lavages performed 5 days after the LH
peak. Five zona-enclosed blastocysts, with an estimated age ranging from
102.5-117 h, were recovered. However, embryos ranging from 2-16-cell stages
were also obtained during a similar time interval. The latter findings suggest that
delayed and arrested embryonic development, which is commonly observed
in vitro, also occurs following in-vivo fertilization and attempted in-situ growth.

Human Reproduction Volume 11 Supplement 1 1996 © European Society for Human Reproduction and Embryology 175
A.Lopata

Based on the above studies it may be concluded that following in-vivo

conception in the human, early blastocysts begin to form during the first few
hours of the fifth day after ovulation (fertilization), and the majority of these
embryos become expanded blastocysts by the middle or end of day 5. Although
hatching blastocysts have not been collected from the uterus, presumably the
human blastocyst sheds its zona during the sixth day of development. It is worth

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pointing out, in passing, that in the non-human primate, such as the marmoset
monkey, hatching uterine blastocysts have been observed, and hatched blastocysts
can be readily recovered from the uterus together with empty zonae containing
a distinct opening.
If the human blastocyst emerges from its zona by the end of day 6, the
trophectoderm would be expected to directly interact with the uterine luminal
epithelium by day 7 after fertilization. It is not known whether the hatched
human blastocyst is capable of initiating implantation immediately after shedding
its zona, or whether the polar trophectoderm, which is the embryonic region that
interacts with uterine epithelium in primates, requires a period of differentiation
before implantation begins. Our studies in the marmoset monkey have shown
that the hatched blastocyst requires a latent period of about 24 h before it attaches
to a substrate such as Matrigel. Available information in the human suggests that
implantation begins on day 7 or 8 after fertilization (Hertig, 1975; Lenton
et al., 1982).
It has been known for a long time that human embryos obtained in an in-vitro
fertilization (IVF) programme will form blastocysts by day 5 of development
(Lopata et al., 1982). A small percentage of such blastocysts are viable since
they have the potential to produce term pregnancies following transfer to the
uterus (Bolton et al., 1991; Menezo et al., 1992; Olivennes et al., 1994). However,
a majority of cultured blastocysts fail to implant probably due to abnormalities
of embryonic growth in vitro. For example, it has been found that human
blastocysts grown in simple culture media contained the following average
number of cells in the trophectoderm and inner cell mass respectively: 39.1 and
19.3 on day 5; 44.7 and 37.6 on day 6; 80.6 and 45.6 on day 7 (Hardy, 1993).
It is likely that human blastocysts of similar age, developing in vivo, would
contain more than double the total number of cells reported above for each stage.
A retarded level of cell proliferation in cultured blastocysts may partly account
for the low implantation rate following their placement in the uterus.
Following natural conception, the human chorionic gonadotrophin (HCG)
secreted by an intrauterine blastocyst could be detected in the serum by day 8
or 9 after the LH peak, in the majority of women studied (Lenton et at., 1982).
This interval probably corresponds to 7 or 8 days after ovulation and fertilization.
It has been proposed that implantation is established by this stage (Lenton et al.,
1982), although its onset occurs a short, but unknown time earlier. In studies on
blastocysts obtained by IVF and embryo culture, HCG released into the medium
becomes detectable by day 7 of development (Lopata and Hay, 1989). The
gonadotrophin is produced by both hatched and zona-enclosed blastocysts at
about the same time of development, suggesting that a pre-programmed timing

176
 Implantation of the human embryo

mechanism is switched on. Attachment of the blastocyst does not appear to be

a prerequisite for the initiation and continuation of HCG secretion. Our studies
on the distribution of mRNA for the (J.- and ~-subunits of chorionic gonadotrophin,
in implantation stage marmoset blastocysts, have revealed that the invasive polar
trophectoderm is the major source of the gonadotrophin (Lopata et aI., 1995).
This region of the primate blastocyst is strategically located for releasing

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trophoblastic hormones into maternal blood vessels during the earliest as well as
more advanced stages of implantation.
It would be of interest to know whether the cells of the inner cell mass play
an inductive role, particularly in transforming the trophectoderm adjacent to them
into an adhesive and invasive phenotype, and also in preferentially switching on
the genes that express the subunits for chorionic gonadotrophin. The biological
value of these series of localized trophoblastic transformations would be that the
inner cell mass, and subsequently the embryonic disc and tissues derived from
it, become closely associated with a region of trophoblast that pursues maternal
blood and hence facilitates the exchange of nutrients and wastes. During this
sequence of events embryonic differentiation and implantation occur at a brisk
rate. By day 9 of development the uterine epithelium is penetrated completely,
syncytial trophoblast becomes well established, and stromal fibroblasts begin to
be transformed into decidual cells. The blastocyst then erodes the basement
membrane, enters the stroma and the trophoblast begins to penetrate maternal
blood vessels by day 10 or 11 after fertilization. At about the same time
trophoblastic lacunae become filled with maternal blood (Rock and Hertig, 1948;
Larsen, 1980). Decidualization, which is not conspicuous during the early stages
of human implantation, becomes more pronounced shortly after day 11.

Clinical aspects of implantation

Complex tissue interactions must be taken into consideration when studying

implantation. These interactions are illustrated in Figures 1-3. A number of
tissue compartments are involved, apart from those present in the blastocyst.
These include the uterine epithelium, and of course its basement membrane
which contains bioactive molecules such as laminin, fibronectin and collagen
type IV. Stimuli essential for implantation are exchanged between the blastocyst
and uterine epithelium, and similar interactions probably also occur between the
blastocyst and decidua, with the epithelium acting as an intermediary (Figure 2).
It is also well known that the decidua is a dynamic tissue composed of many
cell types including haematopoietic cells, reactive blood vessels with properties
specific for the endometrium, and vascular endothelium. Moreover, the cell
population and the composition of the extracellular matrix within the decidua,
vary with its state of differentiation and stage of implantation (Glasser and
McCormack, 1980; Glasser, 1990).
The main clinical problem with implantation is to understand the nature of
the processes which govern the pregnancy rates obtained with IVF and embryo
177
A.Lopata

Corpus Luteum I
Oestrogen + Progesterone
... '.
Stromal Inner Cell
Cells " '"
Laminin .'. Mass

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..... Uterine .....
Decidual
Cells
....
Fibronectin
B
M
Epithelial
Cells
'"
Tropliectoderm
..oL
t
Endothelial Collagen IV
G Iycocalyt<
(Trophoblast)
....
Cells Ceiis
Integrins
Macrophages Proteoglycans
Lymphocytes

Figure 1. Interaction of tissues at implantation. In the embryonic compartment. shown in the box on the
right. the inner cell mass and trophectoderm probably influence each other through paracrine and contact
mechanisms. The polar trophectoderm interacts with uterine epithelium in primates through released signals
such as cytokines and eicosanoids as well as via the components of the glycocalyx and cell-cell attachments.
[ntegrins on the surface of the implanting trophectoderm are subsequently involved in the penetration process
by binding to laminin. fibronectin and other compounds in the epithelial basement membrane and the matrix
of the decidual stroma. The cellular and molecular composition of the stromal compartment. and the capacity
of the epithelium to induce decidualization. arc powerfully influenced by the sex steroids secreted by the
developing corpus luteum. Bidirectional arrows indicate feedback interactions between cellular and matrix
compartments.

transfer. We can produce good blastocysts in vitro, although the implantation

rate is only ~ 10% per embryo. Perhaps we have slightly improved on that figure
in recent years, although only modestly. Of the embryos that do implant, 60%
or more proceed to live births.
Achieving normal implantation is the problem, and low rates could be caused
by various factors. Ovarian hyperstimulation may induce either endometrial or
embryonic problems. I believe that the epithelial layer of the endometrium plays
only a relatively unimportant role, being a kind of passive bystander, during the
process of blastocyst penetration. At best the epithelium may have the ability to
block implantation during some stages of the menstrual cycle. When this inhibitory
influence is removed the epithelium becomes permissive of implantation, but
it is the blastocyst that plays the subsequent active role. It is the polar trophoblast
that intrudes between the epithelial cells, which are pushed aside, and that erodes
the basal lamina and penetrates into the stroma and blood vessels. The duration
of this permissive state has been defined as the implantation window. The
permissive window appears to be tightly regulated in animals such as mice and
rats, but is much more variable in humans, and probably in other primates. Some
investigators have found that the implantation window in the human begins 4-5
days after fertilization and lasts about 4 days Navot et ai., 1989), while others
have claimed that its time-span may even extend up to 8 days (Rogers et ai., 1989).
Based on embryo transfer experiments in animals, it has been assumed that
precise synchrony between embryo and endometrium is important for implantation
178
 Implantation of the human embryo

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1 1
Stromal Transformation

Paracrine Endocrine Matrix

Figure 2. The uterine epithelium appears to be an obligatory intermediate in inducing stromal transformation
(decidualization) during implantation. The epithelium is regarded as being an essential transducer that
delivers the deciduogenic signal(s) to the stroma. The nature of the transmitted epithelial signal(s) are not
known. Physiologically, the initial stimulus arises from the blastocyst, although decidualization can be
induced in the absence of an embryo. The three classes of inductive stimuli from the blastocyst are
illustrated by the arrows. The transformed stroma, in turn, directs cytodifferentiation and function of the
epithelium in contact with it (represented by the paracrine, endocrine and matrix feedback arrows). The sex
steroids have a critical priming role during these tissue interactions.

in the human. If the window of implantation lasts from 4-8 days, beginning on
about day 19 of the cycle, all normal embryos will have formed blastocysts that
have hatched during the receptive interval. Is it possible, therefore, to have
embryo--endometrial asynchrony in women who have an implantation window
spanning 4-8 days? As far as I am aware, there are no studies that correlate the
likelihood of implantation with morphological findings on embryos and uterine
epithelium, within transfer cycles, either at the light or electron microscopic
level. So at present we cannot define normal embryonic and endometrial
morphology, particularly between stimulated cycles, let alone the possible
abnormalities.
It is clear that good quality embryos need to be produced for successful
implantation. Virtually all patients can produce a good endometrium if levels of
oestrogen and progesterone are sufficient without being too high. It follows,
therefore, that any type of ovarian stimulation will produce an epithelium that
will be effective, providing the blastocysts are of sufficiently good quality.
Receptivity can only be induced at the site of the blastocyst within the uterine
cavity. In the absence of an embryo, there is no other way of defining receptivity.
Apart from my belief that the epithelium plays a passive role during blastocyst
penetration, I also consider that there is lack of strong evidence showing that
blastocysts firmly attach to the apical surface of the uterine epithelium. If they
do so, it is a very transitory process, since the blastocyst is very mobile.
In effect, perhaps one of the main roles of the uterine epithelium is to transmit
signals from the blastocyst to induce the decidual cell reaction (Figure 2). A
blastocyst would implant even if uterine epithelium was removed, but it would
not induce a decidual response which is the function of the epithelium. The
implanting blastocyst produces signals, perhaps prostaglandins, leucotrienes,
179
A.Lopata

Lam-Rec
iii'
:::il

. . . . . .L - - -1-
Laminin
---,
J
I:

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Decidual cell Decidual cell

Figure 3. Trophoblast invasion of decidua. Endometrial stromal cells that undergo decidual transformation
acquire the capacity to synthesize and secrete basement membrane proteins, such as laminin, fibronectin and
collagen type IV. The invasive trophoblast of the implanting embryo also expresses and releases some of
these proteins as well as surface receptors (e.g. integrins) for various matrix proteins. Invasive trophoblast
cells have also been found to produce plasminogen activator (u-PA), a serine proteinase that converts
plasminogen to plasmin. High affinity cell slllt'ace receptors (uPA-R) and binding sites for plasminogen and
plasmin serve to localize proteolytic activation to the pericellular area. The plasmin generated is able to
degrade extracellular components and to activate latent metalloproteinases, such as procollagenase. The
proteolytic activity of plasmin is regulated by PA inhibitors (PAI-I and PAI-2) and other proteinase
inhibitors. The events illustrated bring together adhesive and proteolytic interactions during implantation.
E1 = oestradiol:
P4 = progesterone.

platelet activating factor, histamine, or other bioactive agents such as cytokines,

which may play an integrating role (Kennedy, 1983; Alecozay et at., 1991;
Sharkey, 1995) in inducing the decidual cell reaction.
As the blastocyst begins to implant the trophoblast, and particularly invasive
polar trophoblast, produces laminin and fibronectin (Figure 3). At the same time
receptors for these glycoproteins appear on the trophoblast. The appearance of
basement membrane compounds at the apical surface of uterine epithelium, may
play a part in disorganizing the epithelium at the implantation site, in some
species (Foidart et al., 1990). Among other mechanisms, the plasminogen
activator and matrix metalloproteinase systems (Figure 3), are probably involved
in disorganizing the uterine epithelium that interacts with the blastocyst during
the penetration process (Lala and Graham, 1990).
180
 Implantation of the human embryo

The marmoset as a model of human implantation

The marmoset is a very suitable experimental model for human implantation. Its
reproductive cycle, which is normally 28 days, can be controlled by administering
a prostaglandin F2u analogue during the mid-luteal phase or early pregnancy, to
begin a new cycle (Summers et at., 1985). The marmoset ovulates about 8-9

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days after this treatment, and ovulation can be readily detected by plasma
progesterone assays. Since marmosets are very fecund, and are housed as mating
pairs, ovulation nearly always corresponds to day one of pregnancy. Embryos
ranging from the 4-cell to hatched blastocyst stages can be collected from the
uterine cavity during precisely timed fertile cycles, since it is easy to irrigate the
uterine cavity, and flush out the embryos. Cleavage stage embryos can be
successfully grown to hatched blastocysts in a variety of culture media.
One of our models of implantation is to culture marmoset blastocysts on
Matrigel, a basement membrane-like substance. Blastocysts attach to this matrix
in the region of the polar trophectoderm, and subsequently invade it, and since
the penetrating blastocysts retain their three-dimensional integrity the system
provides an excellent model for experimental studies (Lopata et at., 1995). We
have analysed the effects of leukaemia inhibitory factor (LIF) on such blastocysts
around the time of implantation by permitting the embryos to invade the Matrigel
for 4 days in the absence and presence of the cytokine, at various concentrations,
in the culture medium. More cells and mitotic figures were observed in the inner
cell mass of the treated embryos compared with controls. LIF had apparently
accelerated the development of the inner cell mass and the amniotic cavity, but
we could not determine its effect on the endoderm which had formed a complete
yolk sac by day 4 of 'in-vitro implantation'. Our data also suggested that LIF
promoted cytotrophoblast proliferation and to some extent inhibited the formation
of syncytiotrophoblast. These observations remind us that LIF receptors have
been identified on human b1astocysts, and our studies show how some of those
receptors may be expressed on the differentiating tissues of the implanting
primate embryo. The possible LIF interactions during implantation are illustrated
diagrammatically in Figure 4.
Like the human blastocyst, the marmoset (m) blastocyst also produces chorionic
gonadotrophin (CG), and probably other bioactive factors, during early stages of
differentiation (Hearn et at., 1988). The production of mCG interested us because
the gonadotrophin not only rescues the corpora lutea of early pregnancy in
marmosets, but may also have an immunosuppressive action that contributes to
survival of the blastocyst during implantation. There is also clear evidence that
CG stimulates cytotrophoblast growth, so it is probably involved in early placenta
formation, acting as an autocrine growth factor (Yagel et at., 1989).
We have also investigated the localization of CG in the implantation stage
marmoset blastocyst. Bonduelle et al. (1988) reported that mRNA for the HCG
~-subunit is detectable at the 6-8-cell stage human embryo, where every cell is
already transcribing the gene(s). My colleagues at the University of Adelaide
cloned the genes for the u- and ~-subunits of marmoset CG (Simula et al.,
181
A.Lopata

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o (J
,.: , \ ; " "
o o
BM BASAL
Figure 4. Oestrogen induces the synthesis of leukaemia inhibitory factor (LIF) in the uterine epithelium on
the day of implantation. LIF is transported to the blastocyst where it interacts with LIF receptors (LIF-R)
that arc expressed both in the trophectoderm and inner cell mass. mRNA = gene message for LlF-R:
BM = basement membrane.

1995), so we had cDNA available for both subunits. From these sequences, we
obtained riboprobes for the a- and ~-subunits, producing both sense and antisense
probes, which were labelled with digoxygenin for in-situ hybridization. Analyses
involving in-situ hybridization were then carried out on marmoset blastocysts
invading Matrigel4 days after hatching (Lopata et aI., 1995), i.e. early implantation
stages. As a positive control we used marmoset trophoblastic vesicles, which are
known to secrete bioactive CG in tissue culture (Summers et al., 1987; Simula
et al., 1995). Uterus, kidney and liver were used as negative controls.
The mRNA for CG-~ was predominantly expressed over the polar trophoblast
region. Some signals were found in mural trophoblast, but far fewer. Inner cell
mass had a low level of activity of the message for CG-~ in some cells, and
there was virtually no activity in the endoderm.
With the CG-a subunit anti-sense riboprobe, hybridization also occurred over
the polar trophoblast, but not as heavily. The transcript was uniformly distributed
both in polar and mural trophoblast, with some a-subunit activity showing up
distinctly in the inner cell mass. It appears that in the marmoset blastocyst, CG
activity is located predominantly in the area where the trophoblast is attaching
and implanting, whereas in differentiating tissues such as inner cell mass and
endoderm, the genes for the gonadotrophin are probably being inactivated.
182
 Implantation of the human embryo

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