Professional Documents
Culture Documents
com/Article/
www.rbmonline.com/Article/2454 on web 5 October 2006
Outlook
Embryo implantation: the molecular mechanism
remains elusive
John Aplin received his initial training in chemistry at Queen’s College Oxford, and in
biophysics at the University of British Columbia where he first became interested in
glycobiology and the cell surface. As a ‘post-doc’ at the MRC laboratories in Mill Hill,
London his interests turned to the biology of cell adhesion. Chance events led him into
reproductive biology, most notably implantation and placental development. He is currently
Professor of Reproductive Biomedicine at the University of Manchester where he is a
member of the Maternal and Fetal Health Research Centre in the Medical School, with a
cross-appointment in the Faculty of Life Sciences.
Dr John Aplin
JD Aplin
Division of Human Development, Medical School, University of Manchester, UK
Correspondence: Research Floor, St Mary’s Hospital, Manchester M13 0JH, UK; e-mail: john.aplin@manchester.
ac.uk
Abstract
Low rates of implantation are an impediment to more efficient assisted reproduction techniques. Improved endometrial
receptivity and embryo preparation should lead to higher pregnancy rates, lower rates of early pregnancy failure and fewer
multiple pregnancies. As the first site of contact between embryo and endometrium, the luminal epithelium (LE) is responsible
for the non-receptive status of proliferative and early secretory tissue, and transformation to receptivity in the mid-secretory
phase presumably requires alterations in expression, organization or activation of adhesion systems. Luminal cells are less
abundant than their glandular counterparts, and are under-represented in global tissue datasets. Furthermore, alterations in
cell surface composition can be readily accomplished by mechanisms that do not rely on altered transcription or translation.
Current data from in-vitro models are consistent with initial attachment to mucin in the apical glycocalyx, perhaps via a
carbohydrate-mediated interaction, after which the epithelial phenotype is modified by a medium- or short-range embryonic
signal. A cascade of interactions follows, mediating embryo migration across the epithelium. Strikingly, numerous potential
mediators of adhesion at implantation are located in the lateral rather than the apical surface of LE cells. Attached embryos
appear to gain rapid access to this highly adhesive lateral membrane domain.
understanding of the key attributes (molecular or structural) that a primary polarity and an apical barrier. In carcinoma cells, this
confer receptive status on endometrium. In fact, there is evidence tightly defined polarity of MUC1 expression is lost and novel
that a range of phenotypes will support implantation; it may well glycoforms appear. There is evidence that the glycocalyx is
be that healthy embryos can to some extent overcome endometrial altered at the surface of uterodomes in the mid-secretory LE, with
defects. loss of MUC1 epitopes (Horne et al., 2005).
Based on these findings, one can postulate that initial attachment as an adhesion substrate and a barrier. Uterodomes represent a
to glycocalyx is followed by lateral clearance of mucin, leading specialized area of LE plasma membrane that could be used for
to interaction of trophoblast with higher affinity epithelial attachment, but this has not been observed (Bentin-Ley, 2000).
adhesion systems (Figure 1). Thus, the glycocalyx could act both
Figure 1. (a) Initial attachment is to the glycocalyx and may be mediated by a lectin−carbohydrate interaction. The lateral luminal
epithelium (LE) membranes are rich in adhesion molecules. (b) Second phase attachment follows the clearance of apical glycocalyx
in response to an embryonic signal. The lateral LE borders open and trophectodermal processes insert between the LE cells. MUC
1 = mucin 1; OPN = osteopontin.
835
Outlook - Molecular mechanism of embryo implantation - JD Aplin
This initial interaction locates the embryo within a specific epitheliochorial implantation, OPN localizes precisely to the
area of the uterine surface. Short range signalling to maternal interface between trophectoderm and LE (Johnson et al., 2003).
epithelial cells can then occur, leading to glycocalyx clearance. The possibility that OPN could bridge between cell surface
This is a test of viability for the embryo, and poor quality (e.g. ligands such as integrins and CD44 on the trophectoderm and
monosomic) blastocysts may fail at this stage. At the same time, LE has not been tested directly.
it places the state of receptivity in a new light: the ability of
the epithelium to respond to embryo-derived signals (Quenby Integrins of the β1 and αv subfamilies have been widely
et al., 2002). discussed as potential mediators of implantation (see Aplin
and Kimber, 2004). Mice lacking the integrin β1 subunit fail
to implant, but this intervention deletes at least 10 different
Second phase attachment: integrins. In contrast, mice null for αv are fertile. Integrin αvβ3
candidate adhesion systems exhibits a mid-secretory phase increase in expression by virtue
of an increase in β3 abundance (Lessey, 2002). Experimental
First phase attachment is presumed to be followed closely evidence has been reported (blocking antibody action in vivo)
by adhesion of trophectoderm to non-mucin receptors at that suggests a role for integrin α4β1 in mouse implantation
the epithelial surface. There are numerous hurdles to the (Basak et al., 2002). Integrin expression on epithelial cells is
identification of molecular mediators, of which the most serious highly variable between different areas of both GE and LE.
is that it is not possible to obtain sufficient human embryos to In general, expression is substantially higher in the former.
carry out well-controlled studies of molecular pathways. Mouse Integrins of both types are again more abundant on lateral (and
embryos may provide useful clues, and studies of fertility in basal) epithelial surfaces than at the apical surface, though
mouse gene knockouts are in some cases instructive. some apical expression is detectable. Integrins are also widely
expressed on stromal cells.
Candidate adhesion systems have been reviewed in detail
elsewhere (Aplin, 1997; Kimber and Spanswick, 2000; Aplin Trophinin is a membrane component discovered in a screen
and Kimber, 2004). A few are mentioned here in brief. In for components mediating the attachment of teratocarcinoma
assessing possible mediators, the Human Protein Atlas, part of cells to a uterine epithelial cell line (Fukuda et al., 1995).
the Human Proteome Organisation (HUPO) human antibody There is evidence that human chorionic gonadotrophin can
initiative (http://www.proteinatlas.org), is a very useful resource induce local expression of trophinin by maternal epithelial cells
that aims eventually to show the distribution of all proteins in a (Nakayama et al., 2003). Functional trophinin associates with
wide range of human tissues. Several of the proteins discussed two cytoplasmic proteins, tastin and bystin, and the complex
below are included in current releases of the atlas with high appears to be apically displayed (Fukuda and Nozawa, 1999). It
quality immunolocalization in endometrium. is not absolutely required for early implantation in mice (Nadano
et al., 2002), but remains a possible candidate in primates.
Basigin, also known as CD147 and EMMPRIN, is a membrane-
spanning immunoglobulin superfamily member with multiple CD9 is a member of the tetraspanin family of accessory
binding partners at the cell surface. It may be activated by proteins. It can act as a receptor for the family of pregnancy-
direct homotypic interaction between adjacent cells, but is specific glycoproteins (PSG) produced by trophoblast
best known as an activator of MMP activity. In both mice and (Wynne et al., 2006), as well as associating laterally with
rats it is expressed in LE on day 1 of pregnancy under the β1 integrins in the plasma membrane (Park et al., 2000). Its
influence of oestrogen, is down-regulated but then reappears distribution in endometrium is again largely in the lateral
locally in response to an embryonic stimulus on day 4 (Xiao membranes of epithelial cells, including the LE. It is also
et al., 2002a,b). Implantation rates are severely compromised expressed in blastocysts. Blocking antibodies to CD9 added
in animals lacking basigin (Igakura et al., 1998; Kuno et al., in vitro to mouse embryos attaching to epithelial monolayers
1998). Basigin is expressed most prominently at the lateral stimulated trophoblast outgrowth, though there was no effect
epithelial surface of endometrial epithelial cells in human and on attachment rate. MMP-2 production was stimulated via the
rodent. It is also expressed on pre-implantation embryos. phosphatidylinositol 3-kinase pathway. Intrauterine injection of
the antibodies on day 4 of mouse pregnancy increased the number
CD44 is another single pass transmembrane glycoprotein. It of implantation sites (Liu et al., 2006). These experiments
shows a complex pattern of alternative splicing with several suggest CD9 as an inhibitor or regulator of implantation, and in
forms observed in endometrium (Behzad et al., 1994). It agreement with this hypothesis, CD9-replete embryos implant
is expressed in pre-implantation embryos (Campbell et al., normally in CD9-null mothers (Wynne et al., 2006).
1995a). It is associated with cell migration and has numerous
binding partners including hyaluronate and osteopontin. Null
mice are fertile. In endometrium, CD44 shows a predominantly
Regulation of adhesion systems
lateral distribution in both GE and LE (Behzad et al., 1994). that mediate implantation
Osteopontin (OPN; also known as SPP1) is a secreted It is striking that several molecular systems that might potentially
glycoprotein that is regulated by progesterone (Apparao et al., play a role in the epithelial phases of embryo implantation are
2001) and reaches a maximum in the secretory phase of the cycle predominantly localized to the lateral cell membranes of LE
when it immunolocalizes predominantly to the apical cytoplasm cells when studies are carried out in non-conception cycles.
of LE and GE cells. It has multiple binding partners including Furthermore, few show substantial evidence of hormonal
integrins α4β1, αvβ3, αvβ5, αvβ6 and specific splice variants of regulation, either when studied directly at protein level or when
836 CD44 (v3, v6). Null mice are fertile. In ruminants exhibiting large scale transcriptomic screens have been carried out. There
Outlook - Molecular mechanism of embryo implantation - JD Aplin
is compelling evidence that local signalling occurs between the receptor systems be displayed at the apical LE surface in the
embryo and LE at implantation and it is entirely plausible that mid-secretory phase of non-conception cycles. Mouse mutants
such signalling triggers a reorganization of the LE surface with with impaired implantation are useful in hypothesis generation,
alteration of polarity and redistribution of components between but the extent to which embryo attachment in human uses the
membrane domains. Table 1 lists several mechanisms by which same ligand–receptor systems remains to be demonstrated.
alteration in plasma membrane composition or activity could
occur independent of transcription or translation. References
The lateral borders of LE cells undergo striking changes at the Aplin JD 2000 The cell biological basis of human implantation.
time of implantation in several species. For example, desmosomes Baillière’s Best Practice and Research. Clinical Obstetrics and
are reduced in the mouse uterine luminal epithelium during the Gynaecology 14, 757–764.
preimplantation period of pregnancy (Illingworth et al., 2000), Aplin JD 1997 Adhesion molecules in implantation. Reviews of
Reproduction 2, 84–93.
and gap junctional connections between LE cells are exquisitely
Aplin JD, Kimber SJ 2004 Trophoblast-uterine interactions at
modulated by embryonic signals in the implantation chamber implantation. Reproductive Biology and Endocrinology 2, 48.
(Grummer et al., 2004). Tight junctions move to a deeper level Aplin JD, Hey NA, Graham RA 1998 Human endometrial MUC1
in LE in mid-secretory phase human endometrium (Murphy et carries keratan sulfate: characteristic glycoforms in the luminal
al., 1982), and the tight junction proteins claudins 1, 4 and 5 epithelium at receptivity. Glycobiology 8, 269–276.
are concentrated in the lower parts of lateral LE cell borders Apparao KB, Murray MJ, Fritz MA et al. 2001 Osteopontin and its
(http://www.proteinatlas.org). It is noteworthy that when human receptor αvβ3 integrin are coexpressed in the human endometrium
embryos attach to primary human epithelial cells, transmission during the menstrual cycle but regulated differentially. Journal of
Clinical Endocrinology and Metabolism 86, 4991–5000.
electron microscopic examination reveals direct membrane-
Basak S, Dhar R, Das C 2002 Steroids modulate the expression
to-membrane interactions between the trophectoderm and the of alpha4 integrin in mouse blastocysts and uterus during
lateral (not apical) surfaces of endometrial cells (Bentin-Ley, implantation. Biology of Reproduction 66, 1784–1789.
2000). This appears to occur after disruption of apical junctional Behzad F, Seif MW, Campbell S, Aplin JD 1994 Expression of
complexes, with opening up of spaces between the lateral two isoforms of CD44 in human endometrium. Biology of
borders of epithelial cells, and intrusion of trophectodermal Reproduction 51, 739–747.
processes. There is sharing of apical junctional complexes and Bentin-Ley U 2000 Relevance of endometrial pinopodes for human
desmosomes between trophectoderm and LE cells (Lopata et blastocyst implantation. Human Reproduction Supplement 6,
67–73.
al., 2002; Figure 1).
Bergh PA, Navot D 1992 The impact of embryonic development and
endometrial maturity on the timing of implantation. Fertility and
Thus, the interaction between the embryo and the apical LE Sterility 583, 537–542.
surface is transient. Interactions occurring at the highly hydrated Bloor DJ, Metcalfe AD, Rutherford A et al. 2002 Expression of
glycocalyx are difficult to visualize in electron microscopy cell adhesion molecules during human preimplantation embryo
because of dehydration artefacts arising at processing. The store development. Molecular Human Reproduction 8, 237–245.
of cell adhesion molecules in lateral LE membranes may be Buckley CH, Fox H 1989 Biopsy Pathology of the Endometrium.
Chapman and Hall, London.
called into action early in the epithelial phase of implantation,
Cameo P, Srisuparp S, Strakova Z, Fazleabas AT 2004 Chorionic
either following attachment to the glycocalyx or immediately gonadotropin and uterine dialogue in the primate. Reproductive
after its clearance. Biology and Endocrinology 2, 50.
Campbell S, Swann HR, Aplin JD et al. 1995a CD44 is expressed
Returning to the criteria listed at the start of this article, throughout pre-implantation human embryo development. Human
transcriptional regulation of components mediating attachment Reproduction 10, 425–430.
during the menstrual cycle is a questionable assumption. Campbell S, Swann HR, Seif MW et al. 1995b Cell adhesion
Given local signalling at the implantation site, and the many molecules on the oocyte and preimplantation human embryo.
Human Reproduction 10, 1571–1578.
mechanisms available to cells for rapid alteration of cell surface
Carver J, Martin K, Spyropoulou I et al. 2003 An in-vitro model for
composition, neither is it essential that the key adhesion ligand– stromal invasion during implantation of the human blastocyst. 837
Outlook - Molecular mechanism of embryo implantation - JD Aplin
Seif MW, Aplin JD 1990 Cell surface components of human Xiao LJ, Diao HL, Ma XH et al. 2002b Basigin expression and
endometrial epithelium: monoclonal antibody studies. Trophoblast hormonal regulation in the rat uterus during the peri-implantation
Research 4, 339–356. period. Reproduction 124, 219–225.
Talbi S, Hamilton AE, Vo KC et al. 2006 Molecular phenotyping of Zhou XL, Lei ZM, Rao CV 1999 Treatment of human endometrial
human endometrium distinguishes menstrual cycle phases and gland epithelial cells with chorionic gonadotropin/luteinizing
underlying biological processes in normo-ovulatory women. hormone increases the expression of the cyclooxygenase-2 gene.
Endocrinology 147, 1097–1121. Journal of Clinical Endocrinology and Metabolism 84, 3364–
Thathiah A, Carson DD 2004 MT1-MMP mediates MUC1 shedding 3377.
independent of TACE/ADAM17. Biochemical Journal 382,
363–373.
Thathiah A, Blobel CP, Carson DD 2003 Tumor necrosis factor-alpha Paper based on contribution presented at the International
converting enzyme/ADAM 17 mediates MUC1 shedding. Journal Serono Symposium ‘Human implantation: the new frontiers
of Biological Chemistry 278, 3386–3394. of human assisted reproductive technologies’ in Erice, Sicily,
Wynne F, Ball M, McLellan AS et al. 2006 Mouse pregnancy- Italy, May 5–6, 2006.
specific glycoproteins: tissue-specific expression and evidence of
association with maternal vasculature. Reproduction 131, 721–732.
Xiao LJ, Chang H, Ding NZ et al. 2002a Basigin expression and Received 7 June, 2006; refereed 14 July, 2006; accepted 25 August,
hormonal regulation in mouse uterus during the peri-implantation 2006.
period. Molecular Reproductive Development 63, 47–54.
839