Journal of Reproductive Immunology

39 (1998) 117 131

Cytokines and embryo implantation1

Carlos Simon a,b,*, Carlos Moreno a,b, Jose Remoh a,b,

Antonio Pellicer a,b
a
Instituto Valenciano de Infertilidad (IVI), Guardia Ci6il 23, 46020 Valencia, Spain
b
Department of Pediatrics, Obstetrics and Gynecology, Valencia Uni6ersity School of Medicine,
Valencia, Spain

Abstract

The implantation process is currently considered the most relevant limiting factor for
successful pregnancy. It is evident that molecular interactions at the embryo-maternal
interface at the time of implantation are crucial in order to understand the mechanisms of
embryonic implantation. The principal aim of this study is to demonstrate the existence of
specific communication pathways between the human embryo and endometrium. The
molecular interactions appears to be initiated by the endometrium in the presence of an
implanting blastocyst and is mediated through embryonic cytokines (specifically the IL-1
system) and the target is the endometrial epithelial b3 integrin subunit. If the role of b3 is
accepted as a marker of uterine receptivity these results may indicate that the normal
hormonally regulated human endometrium could be the trigger for molecular events prepar-
ing the blastocyst to communicate effectively and regulate endometrial adhesion molecules in
order to implant. 1998 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Cytokines; Implantation; Adhesion molecules; Embryo; Endometrium

1. Introduction

Implantation consists of a series of developmental phases in which the

embryo has to appose and attach itself to the maternal endometrium and
invade into it. The three phases are known as apposition, adhesion and

* Corresponding author. Tel.: +39 96 3624399; fax: + 39 96 3694735; e-mail: @ ivi@futurnet.es

1
Paper presented at the workshop on Paracrine Mechanisms in Female Reproduction, Seville, Spain,
June 1997.

0165-0378/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved.

PII S0165-0378(98)00017-5
118 C. Simon et al. / Journal of Reproducti6e Immunology 39 (1998) 117131

invasion. Apposition is the orientation of the blastocyst within the lumen of

the uterus. Adhesion of the blastocyst is a progressive phenomenon that ties
the embryo to the lumenal epithelium and is the primary event initiating an
embryo-maternal relationship regulated by cytokines and adhesion
molecules (Cross et al., 1994). Invasion is a process that allows the
embryonic trophoblast to penetrate deep into the maternal decidua, invad-
ing the endometrial spiral arteries (Blankenship et al., 1993). The most
intriguing aspect of implantation is that it involves two outstanding players,
the maternal endometrium and the embryo. The communication between
them and the reciprocal effect on each other, is an exciting and as yet
unsolved paradigm in reproductive medicine. Once implantation begins, it
has been estimated that clinical implantation in the human is efficient in no
more than 30% of cases (Miller et al., 1980). The responsibility for this low
implantation efficiency has to be shared between the embryo (30% of
blastocysts are morphologically abnormal at the time of implantation in
vivo; Hertig et al. (1952)) and a defective embryonic-endometrial dialogue
(30% of early pregnancy losses occurs before the expected time of menstru-
ation; Wilcox et al. (1988)). An even higher degree of early embryonic
wastage occurs following assisted reproductive technologies (Simon et al.,
1997b).
A general hypothesis has been postulated by which cytokinesleukemia
inhibitory factor (LIF) and interleukin (IL-1)and their specific receptors,
properly distributed throughout endometrium and embryo and adequately
controlled at the endocrine (hormonal) and paracrine/autocrine (cytokines
and growth factors) levels, may start the mutual recognition of implanting
blastocyst and endometrium (Simon et al., 1996a). Cytokines and growth
factors may also serve as the link in the regulation of molecules that provide
the physical contact between embryo and uterus, referred to as adhesion
molecules (Simon et al., 1996a). As redundancy is one premise to assure the
effectiveness of crucial biological processes, many communication possibili-
ties between embryo and endometrium must exist but unique systems could
also be at play.
The endometrium has traditionally been perceived as a passive organ but
now it is known that it is an active, versatile and hormonally regulated
organ which plays a major role in the process of implantation and in
maintaining a viable pregnancy. It has become evident that human en-
dometrium is an active site for cytokine-growth factor production and
actions (Tabibzadeh, 1991; Tabibzadeh et al., 1995a; Giudice, 1994) and the
embryo is able to communicate with the endometrium using the same
cytokine-growth factor-receptor language (Chard, 1995; Simon et al., 1995b;
Tabibzadeh and Babaknia, 1995b). Recent evidence suggests that the mater-
nal endometrium has to be prepared, in that it is receptive to an implant-
 C. Simon et al. / Journal of Reproducti6e Immunology 39 (1998) 117131 119

ing blastocyst only within a window of implantation, which is temporally

and spatially restricted.
The crucial role of steroid hormones in order to prepare and drive the
endometrium for successful embryonic implantation is beyond any doubt.
However, it is clear that steroid hormones are not final effectors, rather they
may initiate a downstream cascade of molecular events through local
paracrine/autocrine molecules which account for the intimate mechanisms
of receptivity. The expression, regulation and mode of action of several
cytokines, growth factors and adhesion molecules during the implantation
window are just beginning to be understood (for reviews see Giudice, 1994;
Chard, 1995; Lessey and Castelbaum, 1995; Tabibzadeh et al., 1995a;
Tabibzadeh and Babaknia, 1995b). Mouse models have provided insight
into the relevance of some molecules relevant to endometrial preparation.
These effector molecules include members of the EGF family, LIF and
(IL-1).
Heparin-binding EGF (HB-EGF) is a new member of the EGF family
that binds to the EGF receptor acting as a mitogen in several systems. Also,
HB-EGF mRNA has been found to be expressed in human endometrium
throughout to the menstrual cycle (Birdsall et al., 1996).
In mice, HB-EGF is present in luminal epithelium at the site of implanta-
tion before any other sign of blastocyst attachment and disappears when
implantation is delayed by administration of progesterone (Das et al., 1994).
The LIF is expressed throughout the menstrual cycle with significant
increase in the mid- and late secretory phase (Arici et al., 1995). It is
primarily detected in the glandular epithelium in the secretory endometrium
(Charnock-Jones et al., 1994) and is not regulated by steroid hormones
(Arici et al., 1995). However, IL-1, tumour necrosis factor, transforming
growth factor (TGFa) and other cytokines are potent inducers of LIF
expression in endometrial stromal cells. During pregnancy, LIF is expressed
in mouse endometrial epithelium on the 4th day in the presence or absence
(pseudopregnant animals) of an embryo (Stewart, 1994). In fact, blastocyst
implantation depends on the uterine expression of leukaemia inhibitory
factor (LIF) in mice (Stewart et al., 1992), demonstrated by the transgenic
mouse model.
Interleukin-1 (IL-1) is a family of three related polypeptides, two ago-
nists, interleukin-1a (IL-1a) and interleukin-1b (IL-1b) and one inhibitor,
interleukin-1 receptor antagonist (IL-1ra). There are two IL-1 receptors
named IL-1 receptor types I and II (IL-1R tI and IL-1R tII) but IL-1
signalling occurs exclusively via type I receptors. IL-1R tI is expressed at the
mRNA level in the human endometrium throughout the menstrual cycle
(Simon et al., 1993b). More specifically, IL-1R tI mRNA is expressed in the
human endometrial epithelium at increased levels during the luteal phase
120 C. Simon et al. / Journal of Reproducti6e Immunology 39 (1998) 117131

(Simon et al., 1993a). It has been shown that the blockade of endometrial
interleukin-1 receptor type I (IL-1R tI) by its natural antagonist interleukin-
1 receptor antagonist (IL-1ra) prevents implantation in mice (Simon et al.,
1994a) by a direct effect on the endometrial epithelium. However, the
precise role for these cytokines in human endometrium in preparing for
implantation remains undefined.
In humans, the dynamics of the complete IL-1 system, IL-1b (Kauma et
al., 1990; Tabibzadeh and Sun, 1992b; Simon et al., 1993a), IL-1R tI
(Tabibzadeh et al., 1990; Simon et al., 1993a,b) and IL-1ra (Tabibzadeh and
Sun, 1992b; Sahakian et al., 1993; Simon et al., 1995a,b) are known in the
endometrium, endometrial fluid (Simon et al., 1996b), human embryos (De
los Santos et al., 1996) and at the maternal-embryonic interface (Paulesu et
al., 1991; Hu et al., 1992; Librach et al., 1994; Simon et al., 1994c). These
morphological data are in agreement with the demonstrated stimulatory
effect of IL-1 on hCG release from human first trimester trophoblast cells in
culture (Tackacs et al., 1988; Masuhiro et al., 1991; Steele et al., 1992).
The aim of this study is to analyze one specific pathway of molecular
interaction between the human embryo and uterus implicating the embry-
onic IL-1 system. Based on previous and actual studies (Paulesu et al., 1991;
Hu et al., 1992; Sahakian et al., 1993; De los Santos et al., 1996; Simon et
al., 1996b), the hypothesis is that the embryo secretes the complete IL-1
system (IL-1a, IL-1b, IL-1ra) when entered in the endometrial cavity in
response to the presence of endometrial epithelial cells (EEC) (De los Santos
et al., 1996). In turn, the blastocyst acts on the maternal EEC by up-regulat-
ing b3 integrin subunit and this activation is triggered by the binding and
activation of embryonic IL-1a and IL-1b to the endometrial epithelial
IL-1R tI (Simon et al., 1997a). This IL-1 induced-endometrial b3 up-regula-
tion is functionally relevant because it increases the ability of the blastocyst
to adhere to the EEC.

2. Materials and methods

2.1. Experimental design

To investigate the effect of endometrial cells on the regulation and

secretion of the interleukin-1 system by the human embryo the immunohis-
tochemical presence of IL-1b, IL-1ra and the common receptor IL-1R tI in
human oocytes and embryos were first analyzed. Secondly, the IL-1 embry-
onic secretion pattern was evaluated in the conditioned media of embryos
cultured under different conditions. Thirdly, the possibility that an em-
bryoendometrial interaction was involved in the release of these cytokines
 C. Simon et al. / Journal of Reproducti6e Immunology 39 (1998) 117131 121

by the human embryo was investigated by co-culturing individual human

embryos with human endometrial stromal cells (ESC) or with human
endometrial epithelial cells (EEC).
To investigate the effect of single human embryos in regulating b3, a4 and
a1 integrins on human endometrial epithelial cells, a clinical program was
developed in which embryos are co-cultured with endometrial epithelial cells
(EEC) until the blastocyst stage and transferred back to the mother. The
EEC wells were divided according to the embryonic status: (a) EEC with
embryos that reached the blastocyst stage; (b) EEC with arrested embryos;
and EEC without embryos. Under these conditions, these molecules have
been morphologically localized by immunocytochemistry. Second, to quan-
tify the embryonic regulatory effect on EEC monolayers, flow cytometry of
b3, a4 and a1 integrins was performed. Third, the possibility that the
embryonic IL-1 system was involved in the endometrial b3 up-regulation
was investigated by neutralizing experiments and further confirmed by
scanning electron microscopy (SEM) and dose response experiments. Fi-
nally, the functional significance of the endometrial b3 up-regulation in-
duced by the embryo was studied using a mouse embryonic adhesion assay.

2.2. Clinical IVF protocol

The ovarian stimulation protocol using GnRH-a and gonadotrophins has

been previously described (Pellicer et al., 1989). Briefly, a long protocol was
used for pituitary desensitization with administration of leuprolide acetate
(LA), 1 mg/d subcutaneously (Procrin, Abbot S.A., Madrid, Spain), starting
in the luteal phase of the previous cycle. The serum estradiol (E2) levels less
than 60 pg/ml (conversion factor to SI unit, 3.671) and negative vaginal
sonographic scan were used to define ovarian quiescence. The administra-
tion of human menopausal gonadotrophins (Pergonal, Serono, Madrid,
Spain; Fertinorm, Serono, Madrid, Spain) for ovarian stimulation were
monitorized according to serum E2 levels and transvaginal ovarian ultra-
sound scans. The criteria for hCG administration (10000 IU, Profasi,
Serono, Madrid, Spain) were the presence of two or more follicles greater
than 19 mm in greatest diameter and serum E2 levels greater than 800 pg/ml
(2.94 nmol/l). Leuprolide acetate and hMG were discontinued the day of
hCG administration. The oocyte retrieval was performed 3638 h after
hCG administration. The standard IVF procedure has been described
elsewhere (Pellicer et al., 1989). The oocytecumulus complexes (OCC) were
evaluated under the dissecting microscope and classified according to Laufer
et al. (1983). The OCC were incubated at 37C under 5% CO2 in atmo-
spheric air. The embryos were scored on the day of transfer according to
their morphology under the dissecting microscope. A total of four types of
122 C. Simon et al. / Journal of Reproducti6e Immunology 39 (1998) 117131

embryos were classified ranging from type I to IV (Conaghan et al.,

1993). Only embryos type I and II were used for the present study
corresponding to embryos with regular blastomeres without fragmentation
and embryos with regular blastomeres with less than 25% of fragmenta-
tion, respectively.

2.3. Immunohistochemical staining

The immunostaining procedure was performed on human oocytes and

embryos by an avidin-biotin alkaline phosphatase technique as previously
described (De los Santos et al., 1996). They were incubated for 60 min at
37 with the primary antibodies: (a) monoclonal mouse anti-human IL-1R
tI antibody; (b) monoclonal mouse anti-human IL-1b antibody at 20
mg/ml each; (c) polyclonal rabbit anti-human IL-1ra antibody at 20 mg/ml
(all from Genzyme, Cambridge, MA). After rinsing with PBS-T, oocytes
and embryos were incubated with a secondary antibody, biotinylated anti-
mouse IgG (60 min, 10 mg/ml at room temperature) (Vector, Burlingame,
CA) or biotinylated anti-rabbit IgG (60 min, 1.65 mg/ml at room temper-
ature) (Sigma, St. Louis, MO). To amplify the signal, they were incubated
with Vectastain ABC-AP (Vector lab) for 30 min at room temperature.
Control incubations included deletion of the primary antibody.
Endometrial epithelial cells grown on eight-chamber tissue culture slides
(Lab Tek, Miles Scientific, Naperville, IL) were analyzed by indirect im-
munofluorescence (Simon et al., 1997a). Incubated with the primary anti-
bodies: TS2/7 mouse anti-human a1 (1:10000 dilution), B-5410 mouse
anti-human a4 (1:3000 dilution), AP3 mouse anti-human b3 (1:1500 dilu-
tion) each for 60 min at room temperature. After rinsing with PBS-T,
cells were incubated with a secondary antibody: FITC-conjugated goat
anti-mouse IgG whole molecule (60 min, 7.8 mg/ml at room temperature)
(Sigma). Control incubations included omission of the primary antibody.

2.4. Embryo culture

Human embryos at different developmental stages were cultured under

different conditions. A total of three to four human embryos were cul-
tured under oil in 100 ml drops of HAM F-10 (Gibco BRL, Paisley,
Scotland) and 4 mg/ml BSA (Pentex, Miles diagnostic division, Kanka-
kee, IL) (n= 33) or Menezo B2 (Lab CCD, Paris, France) (n =18) or in
wells with 1 ml of Menezo B2 culture media (n=8). The embryo condi-
tioned media (CM) was removed every 24 h for IL-1a, IL-1b and IL-1ra
measurements.
 C. Simon et al. / Journal of Reproducti6e Immunology 39 (1998) 117131 123

2.5. Embryo co-culture

Endometrial samples obtained from secretory endometrium were minced

into small pieces of less than 1 mm and subjected to mild collagenase
digestion. The endometrial stromal cells (ESC) and endometrial epithelial
cells (EEC) were isolated as previously described (Simon et al., 1994b). The
homogeneity of cultures was determined by morphological characteristics
and verified by immunocytochemical localization of cytokeratin, vimentin
and CD68 antigen as described (Simon et al., 1994c). After 6 days, growth
media were replaced by Menezos B2 and the endometrial cells were
co-cultured with single human embryos.
For embryo co-culture, individual human embryos were co-cultured with
experimental EEC and ESC, (n=23 and 4, respectively) for 5 days in 600
ml drops of Menezo B2, starting at day 2 after insemination when they were
at two to four-cell stage and the CM was removed every 24 h. In each
experiment, cultured EEC or ESC in the same volume (600 ml) of Menezo
B2 without any human embryos were used as controls. To further assess the
effect of EEC on the embryonic IL-1 system secretion, human embryos were
cultured with EEC conditioned media (n =9) for 5 days as described (De
los Santos et al., 1996). In this set of experiments, EEC conditioned media
alone was used as a control for each experiment. The IL-1a, IL-1b and
IL-1ra levels (x9 S.E.) were determined by ELISA in the 24 h co-culture
conditioned media. The embryonic developmental stage was defined as the
stage at which the embryo was found on the day of conditioned media
removal. Only embryos type I and II were considered for this study,
degenerated and arrested embryos were also excluded.
In a second set of experiments, individual human embryos were co-cul-
tured with experimental EEC for 5 days (Simon et al., 1997a). The embryos
achieving the blastocyst stage were transferred back to the mother. After
embryo transfer, EEC wells were divided according to the embryonic status
reached: (a) the EEC with embryos that achieved the blastocyst stage; (b)
the EEC with arrested embryos; and (c) the EEC without embryos.

2.6. Flow cytometry

For the flow cytometry (FC) experiments, EEC monolayers were de-
tached by treatment with HBS 1mM EDTA/trypsin EDTA (1/1), washed in
PBS, pH 7.2, centrifuged and the cell pellet was blocked with 1% BSA in
PBS for 90 min at 4C. After washing, cells were incubated with same
antibodies for b3, a4 and a1 as described (Simon et al., 1997a). The data
were expressed as the percentage of stained cells.
124 C. Simon et al. / Journal of Reproducti6e Immunology 39 (1998) 117131

2.7. Neutralizing experiments

The monolayers of the EEC were cultured for 24 h in a pool of

conditioned media from co-cultured blastocysts in the absence (n=8) or
presence of saturating concentrations of anti-hIL-1b (5 mg/500 ml) and
anti-hIL-1a (500 mg/500 ml) (n =4), with saturating doses of anti-hIL-1ra (1
mg/500 ml) (n =4), with recombinant human IL-1ra (10 mg/500 ml) (n =3)
or with blocking doses of anti-LIF (250 mg/500 ml) (n =4) (all from
Genzyme). As negative controls, monolayers of EEC were incubated with
growth media alone. The EEC monolayers were either trypsinized and
analyzed for anti-human b3 by FC or fixed with glutaraldehide 1% for
SEM.

2.8. Dose-response experiments

The EEC monolayers were cultured for 48 h in the presence of increasing

concentrations of IL-1a, IL-1b and IL-1a and IL-1b of 0, 1, 10, 100 and
1000 pg/ml (Simon et al., 1997a).

2.9. ELISA

The IL-1a, IL-1b and IL-1ra concentrations were measured in the

conditioned media from dose-response experiments using a kit from R and
Systems (Minneapolis, MN) (De los Santos et al., 1996; Simon et al.,
1997a). The sensitivity was 0.2, 0.3 and 22 pg/ml, respectively. The intra-
and interassay coefficients of variation were 3.2 and 4.3%, 3.4 and 5.1%,
and 5.2 and 5.3%, respectively.

2.10. Mouse embryonic adhesion assay

To analyze the adhesion enhancement in a mouse model blastocysts were

flushed from the uteri on day 3.5 of pregnancy from PMSG/hCG stimulated
8-week-old Swiss females (CFLP) that were mated and plugged with males
of the same strain and age. The mouse blastocysts thereby retrieved were
rinsed and placed in human EEC wells, divided according to the experimen-
tal design in EEC previously co-cultured with human embryos that achieved
the blastocyst stage, EEC co-cultured with arrested human embryos and
EEC without human embryos. Between two and five mouse blastocysts were
cultured per EEC monolayer at 37C in a 5% CO2, 95% air-humidified
incubator. The percentage of blastocysts attached to the EEC monolayer
was recorded after a 48 h incubation period. To identify embryo adhesion,
a small amount of medium was gently flushed on each embryo by a glass
 C. Simon et al. / Journal of Reproducti6e Immunology 39 (1998) 117131 125

pipette as described by Austgulen et al., 1995. Briefly, embryos that showed

no movement while being observed under an Olympus inverted phase-con-
trast microscope were considered to be attached.

2.11. Statistical analysis

The percentage of cells stained was expressed as mean 9S.E.M. For

statistical comparison among groups, Chi-square or analysis of variance
(ANOVA) followed by Fisher PLSD test were applied; PB0.05 was
considered statistically significant.

3. Results

3.1. Embryonic response to the maternal endometrium

To investigate the potential role of the interleukin-1 system in the human

embryo the presence of immunoreactive IL-1b, IL-1ra and the common
receptor IL-1R tI were first localized during the preimplantation period.
Immunostaining for IL-1b, IL-ra and IL-1Rt-I was confirmed in oocytes
and embryos in all developmental stages analyzed. The human oocyte
cytoplasm and plasma membrane stained positive for IL-1b, IL-1ra and
IL-R tI, whereas the polar body did not show any staining for IL-1b and
attached granulosa cumulus cells were negative for IL-1ra. During embry-
onic development, IL-1b, IL-1ra and IL-R tI staining was confined to the
cytoplasm and plasma membrane with no statistical differences between
different developmental stages.
The IL-1a and IL-1b were absent in conditioned media (CM) of embryos
cultured either in 100 ml drops under oil of HAM F-10 and 4 mg/ml BSA,
Menezo B2 or 1 ml of Menezo B2 at all developmental stages analyzed. The
only exception was that detectable levels of these cytokines were found in
three out of five conditioned media obtained 24 h after insemination of
oocytes.
With the knowledge that the immunoreactive protein was present, the
attention was focused on the possibility that human endometrial factors
might be involved in the release of these cytokines by the human embryo.
The IL-1a, IL-1b and IL-1ra were absent in CM of embryos co-cultured
with ESC and their controls. However, when single human embryos were
co-cultured with endometrial epithelial cells (EEC), two different popula-
tions of embryos were observed: (a) the IL-1 producers, described as
embryos producing IL-1a and/or IL-1b; and (b) the IL-1 non-producers,
those on which neither IL-1a nor IL-1-b was detected. To confirm the
126 C. Simon et al. / Journal of Reproducti6e Immunology 39 (1998) 117131

positive effect of endometrial epithelial cells on the embryonic IL-1 sys-

tem secretion, human embryos were cultured with endometrial epithelial
cells conditioned media alone showing a similar pattern as described
before. The IL-1a-IL-1b and IL-1ra secretion patterns were further ob-
tained from the IL-1 producer population of single human embryos co-
cultured with EEC during the embryonic development. The
concentrations (mean9 S.E.) of these cytokines were 1.590.4, 0.489
0.21 and 137.1 942.9 for IL-1a-IL-1b and IL-1ra, respectively. Values
were obtained after subtraction of the levels from controls performed
each day from the values obtained in the co-culture. No differences
were found in IL-1a-IL-1b and IL-1ra levels during embryonic develop-
ment. However, the ratio IL-1a-IL-1b/IL-1ra at the blastocyst stage was
significantly lower compared to the ratios at the six-cell and four-cell
stages.

3.2. Embryonic effect on the maternal endometrium

To investigate the potential embryonic regulation of endometrial inte-

grins the presence of immunoreactive b3, a4 and a1 were localized in
cultured human EEC. After embryo transfer, the EEC wells were di-
vided according to the embryonic status reached: (a) EEC with embryos
that achieved the blastocyst stage; (b) EEC with arrested embryos; and
(c) EEC without embryos. Immunostaining for b3 was positive in
plasma membrane of the EEC with increased intensity in wells from
embryos that reached blastocyst stage compared to those EEC wells
from arrested embryos. The FC showed a mean percentage of b3
stained cells of 24.195.7 in EEC co-cultured with embryos that
achieved the blastocyst stage (n=13) versus 9.591.6 (PB0.05) in those
EEC cultured with arrested embryos (n=12). Immunostaining for a1
and a4 integrins was negative in the EEC monolayers studied, regardless
of the presence or absence of embryos and these findings were confi-
rmed by FC. The possibility that the embryonic IL-1 system was in-
volved in the endometrial b3 up-regulation was investigated by
neutralizing experiments demonstrating a significant inhibition of b3
stained cells when EEC monolayers were cultured in the presence of
EEC/blastocyst-conditioned media with (n=4) versus without (n= 8)
anti-human IL-1a and IL-1b (1.65 vs. 14.6%, PB0.05). The dose re-
sponse experiments further demonstrated an up-regulation of b3 positive
cells when IL-1a and IL-1b were added to the medium at a concentra-
tion of 10 pg/ml compared to control medium without added cytokines
(40 vs. 20%, n= 4). The functional relevance of the EEC b3 up-regula-
 C. Simon et al. / Journal of Reproducti6e Immunology 39 (1998) 117131 127

tion was tested using a mouse blastocyst adhesion assay. More mouse
blastocysts attached to EEC previously in contact with human blastocysts
(72.7%) compared to those EEC previously in contact with arrested em-
bryos (40%) (Simon et al., 1997a).

4. Discussion

The results reported herein demonstrate the existence of a specific

molecular cross-talk between the human embryo and endometrium. This
pathway of molecular interactions seems to be initiated by the
endometrium in the presence of an implanting blastocyst; it is mediated
at least in part by embryonic cytokines such as the IL-1 system and the
target is the up-regulation of endometrial epithelial b3 integrin. If the
role of b3 as a marker of uterine receptivity is accepted (Tabibzadeh,
1992a; Lessey et al., 1992, 1994), these results may indicate that the
normal hormonally regulated human endometrium could be the
trigger for molecular events preparing the blastocyst to comunicate effi-
ciently and regulate endometrial adhesion molecules in order to im-
plant.
Based on this work, a model has been proposed to understand the
molecular mechanisms underlying the adhesion of the blastocyst to the
endometrial epithelium. In response to an unknown endometrial epithe-
lial factor, the human blastocyst is able to secrete the complete IL-1
system (IL-1a, IL-1b/ IL-1ra) which is constitutively present but
not secreted, as demonstrated by the selective release in the presence of
EEC (De los Santos et al., 1996). The binding and activation of embry-
onic IL-1a and IL-1b to the endometrial epithelium induces an up-regu-
lation of b3 subunit (Simon et al., 1997a). The endometrial b3
up-regulation is functionally relevant because it increases the ability of
the blastocyst to adhere to the EEC monolayers (Simon et al., 1997a)
(Fig. 1).
The purpose of this research was to gain knowledge about the physio-
logical human embryonic IL-1 secretion pattern. Based on this and pre-
vious studies (Tackacs et al., 1988; Kauma et al., 1990; Tabibzadeh et
al., 1990, 1989; Tabibzadeh and Sun, 1992b; Sahakian et al., 1993; Si-
mon et al., 1993a,b, 1994a,b,c, 1995a,b), it was hypothesized that the
balance between the endometrial and the embryonic IL-1 system (IL-1a,
IL-1b and IL-1ra) must be relevant for embryonic implantation.
This model provides evidence that the endometrium triggers a mecha-
nism whereby the human blastocyst modulates human endometrial re-
ceptivity.
128 C. Simon et al. / Journal of Reproducti6e Immunology 39 (1998) 117131

Fig. 1. Molecular mechanisms underlying the adhesion of the blastocyst to the endometrial
epithelium. In response to an unknown endometrial ephitelial factor (discontinuos arrows),
the human blastocysts is able to secrete the complete IL-1 system in presence of EEC. The
binding and activation of embryonic cytokines (IL-a and IL-b) to the endometrial ephitelium
induces an up-regulation of b3 subunit. Endometrial b3 up-regulation is functionally impor-
tant because it increases the ability of blastocyst to adhere to EEC monolayer.

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