Key words :blastocyst, implantation, placentation, endometrium, paracrine factors, decidualization, endocrine factors, receptivity, NK cells, primary cell culture
Models in experimental biology
Experimental models for human implantation
Window of implantation
Embryo-endometrium dialogue and endometrial receptivity
for blastocyst implantation
Endocrinology of blastocyst implantation
Paracrinology of blastocyst implantation
Endocrine and paracrine factors during implantation
Inflammation paradigm of blastocyst implantation
vitro model of blastocyst implantation
of the cellular and molecular basis of blastocyst implantation
in the human is an intriguing and complex endeavour. Temporal
and spatial integration between embryo and endometrium is
necessary to initiate their interaction in processes leading
to implantation and placentation. It is believed that an
understanding of such cellular events will help to promote
methods for the treatment of infertility, and for developing
newer approaches for safe, effective and acceptable methods
of fertility regulation and reproductive health care.
in experimental biology
understand the physiology of' a given biological behaviour,
very often traditional control engineering systems are articulated
into logical patterns of relationships. In our effort to
study control and regulation, we attempt to assign some configuration
of relationship in the form of a model. Thus, a model can
be viewed as the basis for one or more testable concepts towards
understanding and using physiology of a given behaviour.
The homeodynamics of a model system should be cognisant of
the noise-signal and non-linearity components in the set of
complex interactions among obligatory and facultative biological
events of varying depths in time and space (Fig. 1).
for full view
1 : Kuhn's model of the process involved in the paradigm
shift in the structure of scientific revolution. According
to Thomas Kuhn, observations using models (M) to test
a given hypothesis (H) under a paradigm (P) form clusters
of results (shaded cubes) which often and generally
support the paradigm. However, with time there occurs
an increasing number of outlying observations (conical
projections from coloured cubes) and eventually
they result in a 'pre-paradigm shift revolution' initially
perceived only by a small group of observers. In this
process, an altered awareness takes place, which results
in a paradigm shift. (Kuhn T. The structure of scientific
revolution. Chicago: University
of Chicago Press 1970).
models for human implantation
the Ward Burdick Award Address, Hertig (1) had made a plea
that in order to understand the abnormal there is a need to
study the normal! He then went on to describe the 15 year
saga of his experiences with John Rock in the collection of
normal human embryos and endometrium following hysterectomy
of proven fertile women before the first missed menstrual
period. These studies provided the basic foundation of normal
and abnormal human embryo development during the very early
stages of gestation and led to the concept of gestational
endometrial hyperplasia (2). The dedication of Hertig and
his colleagues led to establishment of the first experimental
model of human implantation. The bioethics that govern science
and scientific studies today, however, does not permit the
use of human materials for studies on implantation. Several
models nevertheless have been developed using human and nonhuman
primate species and these will be elaborated in the present
The idea that endometrium exhibits a particular 'window' in
its pattern of receptivity for blastocyst implantation arose
from experiments using a number of laboratory animal species
(3). However, it was necessary to test this hypothesis using
the primate, especially in view of the fact the high rates
of failures are experienced in conception rates following
transfer of in vitro fertilized embryos to host uterus
(4). Bergh and Navot (5) proposed a window of implantation
in the human which is characterized by endometrial characteristics
observed between cycle days 20 and 24 based on retrospective
analysis of human chorionic gonadotrophin (hCG) in serum of
women who underwent embryo transfers and thereby they helped
to establish a testable model in the human. The biochemical
principles evaluated by histological, immunocytochemical,
in-situ hybridization, Western and Northern blots and
gene array expressions are now the subject of scrutiny by
investigators in their quest of evaluating endometrial receptivity
from endometrial biopsies collected during the proposed 'window'
of healthy volunteers during normal menstrual cycle and from
women experiencing either absolute or relative implantation
dialogue and endometrial receptivity for blastocyst implantation
The potential role
and the involvement of a fertilized embryo in the events leading
to blastocyst implantation, however, cannot be overlooked.
We had rationalized that functional differentiation of' endometrium
can occur only in the presence of a preimplantation stage
embryo in the reproductive tract and thereby pre- and peri-implantation
events cannot be mimicked in endometrium by mid-luteal phase
tissue of non-conception cycle (6). This hypothesis was tested
using the Rhesus monkey as an experimental anima. Timed post-ovulatory
stage endometrial tissue samples from non-conception cycles
and from potential conception cycles were collected along
with the synchronous retrieval of preimplantion stage embryo
(Fig. 2). Though the levels of estrogen and progesterone
in circulation are unchanged in mid-luteal stage of non-conception
and conception cycles, morphometric and biochemical evidence
(7-8) revealed that luteal phase endometrium functionalis
in the presence of a preimplantation stage embryo show differential
changes commensurate with induction of its ‘receptive status’
for blastocyst implantation. Further more, a ‘switching off’
of lysosomal enzyme machinery which in a non-coception cycle
results in results in menstruation was also reported (9).
The observed morphological characteristics in receptive stage
of conception cycles (Fig. 3) indicated a marginal delay of
about 2 days with increased epithelial mitoses on day 6 of
conception (7) and this fitted well with the reported delay
in maturation of human endometrium collected from proven conception
cycles at a later time of gestation (10).
for full view
2: An experimental model to study the nature of embryo-endometrial
interaction leading to the induction of receptive stage
endometrium in the Rhesus monkey.
for full view
3. Transmission electron micrographs of receptive stage
endometrial functionalis collected on day 6 post-ovulation
of proven conception cycles of the Rhesus monkey based
on the retrieval of age- and stage matched preimplantation
stage embryos. (A, B) Endometrial granulated lymphocyte
(arrow) also known as natural killer- like cell
shows close contact with stromal- predecidual cell (arrow-head).
The endometrial granulated lymphocyte is characterized
by kidney-shaped nucleus located eccentrically and membrane-bound
dense granules (g) and shows close apposition with the
plasma membrane of pre-decidual cell having large numbers
of rough endoplasmic reticulum with interconnected cisternae
enclosing dense amorphous material(*). Pre-decidual
cell shows characteristic thickening of inner leaflet
of its plasma membrane and accumulation of pleomorphic
granules (thin arrow) in the inter-membranous
zone. [C] Endothelial cells with cytoplasmic protrusions
(arrow-head) into lumen and presence of transcytotic
vesicles in a capillary lying within an edematous matrix.
Bars : 4 mm
(A), 2.5 mm (B), and 6.5 mm
about the need for a dialogue between a growing preimplantation
stage conceptus and mid-secretory stage endometrium as a pre-requisite
for endometrial receptivity for blastocyst implantation in
the primate has thus resulted in a paradigm shift in the model
structure for studies on nidation (Fig. 4).
for full view
4. Model ofembryo-endometrial dialogue involving secretory
signals from both compartments. B, blastocyst. Ep, epithelial
cells. Bm, basement
ECM, extra-cellular matrix. F, fibroblast. Gl, gland.
V, blood vessel.
of blastocyst implantation
of sex steroid hormone requirement for blastocyst implantation
is well documented based on studies using several non-primate
and primate species. Using the Rhesus monkey as an experimental
animal we have tested whether luteal phase ovarian estrogen
is essential for supporting embryo implantation. Our results,
based on experimental transfer of embryos at morula and blastocyst
stages to surrogate host oviduct of animals who had undergone
bilateral ovariectomy immediately following embryo transfer,
or in monkeys who had been long-term ovariectomized and then
primed with steroid hormones, led us to conclude that ovarian
estradiol is permissive, but not essential for implantation
in mammals that do not undergo diapause (Fig. 5; 11-14).
Similar results were also reported for agonadal women undergoing
IVF-ET with supportive hormonal therapy (15). The observation
that secretary maturation of endometrium occurs with progesterone
alone, without any estrogen in the primed uterus in the human
(16) and in the Rhesus monkey (11) gives us clear indication
thit progesterone alone may support implantation in these
species. Using mifepristone is a tool to inhibit the cellular
action of progesterone, the critical need for progesterone
in establishing early luteal phase receptivity for blastocyst
implantation (Fig. 6) has also been established in the human
(17) and in the Rhesus monkey (18).
for full view
5: An experimental model to study the requirement of
ovarian estrogen and progesterone for blastocyst implantation
in the primate using
and age-matched transfer of pre- implantation stage
morula and blastocyst to oviduct of long-term ovariectomized
surrogate monkeys primed with estrogen (E) and estrogen
+ progesterone (E+P) or progesterone (P) alone in the
secretory phase of simulated cycle.
for full view
6: Experimental models in the human and the Rhesus monkey
to investigate the actions of luteal phase progesterone
in blastocyst implantation.
of blastocyst implantation
stage human endometrium show morphologic evidence of close
interactions between epithelial and stromal cells (19) supporting
the tenet that epithelial cell renewal and differentiation
are tightly regulated through signals derived from stromal
cells to meet the functional demands during reproductive cycle
(20). Estradiol and progesterone are regulators of cyclical
function and they act via their respective receptors to activate
the transcription of target genes. During the window of implantation,
endometrial glandular epithelial cells lack receptors for
both estrogen and progesterone, but progesterone receptors
are detected in stromal cells (21). A 24 kDa beat shock
protein (hsp) has been shown to be maximally expressed in
luminal epithelium of human endometrium around the time of
implantation (22), and steroid
regulated expression of inducible form of hsp has also been
reported in human endometrium in epithelial, stromal, endothelial
cells and in lymphoid aggregates (23). It has been suggested
that hsp 70 expression in human endometrium may play a role
in regulating cell proliferation and/ or down regulation of
sex steroid receptors especially in glandular cells (24).
subset of endometrial samples classified as normal based on
histology have been found to be abnormal on the basis of their
protein expression (25). Progesterone is known to regulate
the synthesis and secretion of a number of proteins (26) including
placental protein 14 (27). The likely role of PP14 and leukemia
inhibitory factor (LIF) showing timed expressions in mid-secretory
stage endometrium in human implantation process has been suggested
based on the data available from infertile women (28-29).
The importance of cytokines such as leukemia inhibitory factor
(LIF), vascular endothelial growth Factor (VEGF), transforming
growth factors (TGFs), tumour necrosis factors (TNFS) and
other biomolecules such as PP14, prostaglandins (PGs) and
several of the matrix metalloproteineases (MMPS) as autocrine
and paracrine factors regulating, implantation cellular and
molecular events has been addressed elsewhere (30).
Endometrial epithelium is known to play a major role in determining
its receptive status for an embryo since its removal from
non-progestational uterus overcomes the block to implantation
normally seen under this condition (31). Recent evidence
suggests that plasma membrane transformations make epithelial
cells less firmly attached to each other and to the extracellular
environment to favour the implantation process (32). The
modulation of tight junctions and or re-modelling of adherens
junctions with differential distribution of E-cadherin/plakoglobin
complexes and E-cadherin/beta catenin complexes may be correlated
with the development of apical adhesiveness of human uterine
cells (33). Cell adhesion molecules involved in cell-cell
and cell-matrix interactions have been recognized to contribute
to cell migration, matrix organization and transduction of
differentiation signals (34). The coexpression of avb3 and a4b1 in human endometrium during the 'implantation
window' has been documented, and the lack avb3 in luteal phase deficiency, endometriosis and infertility are consistent
with the suggestion that these integrins are involved in the
implantation process (35-36).
Temporal and spatial expression of pinopodes on luminal surfaces
led to the speculation that pinopode formation may define
development of uterine receptivity for blastocyst implantation
(37). Fine structural, cell biological and molecular biological
studies of initial apposition and adhesion of trophoblast
to endometrium have not yet been undertaken in primate species.
In the existing literature no single report is available describing
the adhesion stage of primate embryos, however, blastocysts
recovered on estimated postovulation day 8 show significant
degree of cytoplasmic projections which may play a role in
blastocyst orientation and adhesion (38). Luminal epithelial
cells of human and non-human primate species are known to
express a variety of glycoconjugates with usual and unusual
oligosaccharide structures such as PP14, LIF, Lewis Y antigen
and CD 44, which have also been implicated in endometrial
receptivity (39). Progesterone stimulates de novo
synthesis of glycans but their secretion appears to occur
via progesterone-independent intracellular pathways (40).
The expression of mucin molecule MUC-1 is up-regulated in
secretary phase of human endometrial cells (41), and may also
be selectively downregulated at the site of trophoblast-epithelial
apposition through paracrine mechanisms requiring embryonic
signals to trigger the removal of the mucin barrier (42).
Studies in murine species and in the human lend support to
the concept that autocrine control mechanisms involving endocrine-cytokines
operate in a stage specific manner to regulate embryo growth
and differentiation during the cleavage stages (43-44). Transcripts
for gonadotrophin releasing hormone (GnRH) and GnRH receptor
have been detected in human embryos (45) and transcripts for
human chorionic gonadotrophin (hCG) detected it the 2 cell
stage though secreted hCG protein is detectable in culture
media only at days 7-8 post-ovulation (46). Blastocysts of
a number of species including the human can metabolize steroids
and possess the ability to secrete estradiol-17b into the surrounding medium (47).
Early cleavage stage, morula and blastocyst stage human embryos
can secrete PGE but not PGF2a (48), while
in the Rhesus monkey, PGF2 but not PGF2a was detected in the culture medium
of embryos only at the blastocyst stage of development (49).
Using two dimensional gel electrophoresis, we have recently
documented the ability of single Rhesus monkey embryo to synthesize
and to secrete de novo stage-specific proteins only
at blastocyst stage, such secretary functions were not found
in pre-blastocyst stage embryos or in embryos exhibiting,
gross morphological abnormality and/or asynchronous development
(50). Altered profiles of differentiation in cells of inner
cells mass (ICM) and in trophectoderm at blastocyst stage
of development are observed from studies in which gene methylation
was observed only in cells of ICM (51), the ability to secrete
hCG by syncytiotrophoblast cells (46), co-expression of Gal-1
and Gal-3 in abembryonic murine trophectodermal cells (52)
coupled with down regulation of Leptin and STAT 3 in ICM and
their expression by trophectoderm of human embryos (53).
It is likely that such differential functions of ICM and trophectoderm
may provide a blastocyst with the cellular machinery to initiate
a 'dialogue' with maternal uterine cells. Indeed it is reported
that the transfer of human blastocyst to host uterus results
in better rates of implantation compared with early stage
embryo transfers (54).
While the nature of an endometrial 'dialogue'
with pre-implantation stage embryo is still under investigation,
studies in the Rhesus model reveal that systemic inhibition
of' progesterone action in endometrial cells with resultant
endometrial dysfunction and luminal insufficiency (55) resulted
in arrest of embryo development at morula stage and loss of
embryo viability (Fig. 7; 56-57). The critical need of enibryotrophic
secretary factors of endometrial origin is substantiated from
the current clinical experience of co-culturing human embryos
in IVF-ET practice (58).
for full view
7: Ultrastrlictural clinructoristies otpre-implantatioii
stage blastoeysts recovered on day 6 poat-ovulatmn from
uteri of monkeys exposed either to vehicle (A, B; benzyl
ben zoato; olive oil: I ; 4; v/v) or mifepriatoiie (C,
D; 3 rag/kg body weight in above-mentioned vehicle)
i.m. (A, B) Polar trophoblast cells show extensive junetional
complexes (arrow-head), numerous imtoebondria
are present in polar trophoblast cells and with a cytoplasmie
proJection ofendodermal cell.(C, D) Blastomeres from
an embryo recovered from mifepristone-exposed monkey
show numerous degenerative features such as presence
of myelin bodies (), multivesicular bodies (m), lipid
droplets, lipofuscin bodies (lb) and lysosomes (1),
typical karyorrhexis (k). Bars: 1 mm (B), 10 mm (C), and 10 mm
(D) (Reprinted from Ref. 57).
and paracrine factors during implantation and placentation
The contributions of Hertig, Rock and
Adams provide the essential foundation to
the morphological basis of embryo-uterus interaction during
the early stages of placentation in the human (59). The excitement
of finding and describing, the early stages of human ova and
implantation are clearly discernible and palpable from the
available documents. This unique and rare study visualizing
the morphological adaptations during the very early stages
of implantation in the human are now available in the Carnegie
collection of the National Museum of Health and Medicine in
It is interesting to note that Hertig and Rock had employed
the experimental technique first devised by Heuser for dissecting
monkey uteri to obtain early embryos (60). Using timed gestational
stages, Hendrickx (61) used a similar model to document the
morphological basis of preimplantation embryo development
and implantation in the baboon, and Allen Enders and his colleagues
have described the ultrastructural characteristics of preimplantation
stage embryo development, differentiation and trophoblast-uterine
interactions using several macaque species (62-63). In the
macaque, stage-specific endometrial responses to trophoblast
penetration involve highly timed and sequential series of
events beginning with onset of sub-epithelial edema, differentiation
of epitbelial cells into plaque acini followed by decidualization
(64). In rodents, decidualization occurs as an imniediate
response to trophoblast-epithelial interaction at implantation
following the transduction of embryonic signals to epithelial
and stromal cells (65). However, in the macaque and in the
baboon, decidualization is initiated a few days after trophoblast
invasion and at a time when plaque acinar degeneration is
initiated and placental villi begin to form. The nature of
embryonic cues if any for stromal cell decidualization remain
to be explored. However, as will be discussed later, the
cellular and temporal aspects of endometrial decidual cell
responses can also be mimicked by providing an artificial
physical stimulus to appropriately hormone primed uterus.
The early events in embryo-uterine interaction
leading to lacunar and villous stages of placentation have
now been clearly documented in human and macaque (Fig. 8).
The nature of endocrine-paracrine mechanisms regulating trophoblast
invasion into epithelial, stromal and vascular compartments
of endometrium and associated cell-matrix re-modelling, angiogenesis
and hyperplasia that occur in maternal endometrium during
implantation-placeiitation now under critical review based
on the Hertig-Rock experimental model in nonhuman primate
species (Fig. 9; 38).
for full view
8: Human implantation stage 5a at trophoblast
plate stage (A), and stage 5b at lacunar stage
(B) taken from the Carnegie collection. Trophoblast
cells (arrow) are near to, but have not penetrated
maternal blood vessels (A), and initial stage of vascular
invasion observed in lacunar stage (B). Rhesus monkey
lacunar (C, E) and early villous (D, F) stages of implantation.
Trophoblast cells identified from their immunopositive
cytokoratin staining show initial stage of infiltration
into maternal blood vessel (arrow) in lacunar
stage (C) and by early villous
have occupied one wall of maternal blood vessel (arrow-head}
at base of cytotrophoblast cell columns (CC) (D). Stromal
cells and extra-embryonic mesenchymal cells are identified
from their immunopositive staining for vimentin (E,
F). Bars; 30 mm (A, B) and
60 mm (C-F).
for full view
9: Experimental model for collection of timed gestational
stage tissue from mated Rhesus monkeys for the study
of stage-specific gene and
expressions and morphology.
Experimental evidence suggests that
estrogen plays a minimal role at the site of foeto-trophoblast
interaction in the primate (66). Down regulation of' estrogen
activated protoncogenes, cfos and cjun both members of the
Ap-1 transcription factor complex in human decidua (67) lends
further support to the notion that estrogen plays a minimal
role if any during implantation and placentation in the primate.
The involvement of growth factors such
as insulin like growth factors I and II (IGF I and IGF II)
during the early stages of pregnancy suggests a role of IGF
II as an essential survival factor (68) in lamellar syncytiotrophoblast
cells in their transition from proliferating to a differentiated
state (69). IGF II found in trophoblast cells within the
chorionic plate during lacunar and early villous stages may
potentiate their invasive behaviour (70) and stimulate insulin
like growth factor binding protein-I (IGFBP-1) production
by decidual cells in a paracrine manner (71). IGFBP-1 can
in turn influence integrin mediated migration of cytotrophoblast
cells into maternal stroma (72).
or excessive trophoblast invasion can result in complications
of pregnancy such is early spontaneous miscarriage, recurrent
or spontaneous abortions, preeclampsia and fetal growth retardation
of vascular origin in case of, defects associated with placenta
accreta or percreta. Implantation-placentation coordinated
angiogenesis and trophoblast outgrowth and it is important
to understand the development of the vascular network in the
relationship to trophoblast functions. Temporo-spatial distribution
of vascular endothelial growth factor (VEGF) and placental
growth factor (PlGF) his been documented based on I hybridization
and immunohistochemical studies (73). A relatively higher
level of VEGF expression in migrating trophoblast cells in
lacunar stage suggests that hypoxia may function as a stress
factor in inducing VEGF expression (74) and either directly
or indirectly influences endometrial perfusion, proliferation
and villous formation (75). The activation of constitutive
nitric oxide synthase (NOS) by VEGF in human endothelial cells
(76) may further contribute to their angiogenic properties;
Angiopoietin 1 and 2 (Ang-1, Ang-2) secretory angiogenic growth
factors bind and induce tyrosine phosphorylation of Tunica
interna endothelial cell kinase (Tie-2) and their receptor
have been co-localized in first trimester human placenta (77).
It has been suggested that Ang-2 stimulates an increase in
trophoblast DNA synthesis and the release of NO, whereas Ang-1
acts as a potent chemotactic factor for trophoblasts.
paradigm of blastocyst implantation
Decidual cell response is integral to
implantation-placentation response of ednometrium. In primates,
decidual transformation of stromal cells is accompanied by
the recruitment of a special type of lymphocyte known as endometral
large granulated lymphocyte which is a type of natural killer
cell (NK). In conception cycles, NK cells are found in close
contact with stromal cells (Fig. 2), and it has been suggested
that NK cells may play a critical role in influencing mid-secretory
stage endometrium towards making the decision of either to
decidualize in the presence of' viable blastocyst, or to undergo
menstruation (78). Interestingly, mid- to late-luteal phase
primate endometrium employs tools for acute tissue inflammation,
namely pro- and anti-inflammatory agents, albeit in a differential
ratio for both processes : receptivity-blastocyst implantation
and menstruation. NK cells possess receptors for trophoblast
HLA class I molecules and may influence trophoblast migration
(79). The role of uterine NK cells and macrophages in determining
the inflammatory paradigm of implantation (Fig. 10; 80) could
be explored in an experimental artificial model of decidualization
(Fig. 11) in which endometrial responses to a deciduogenic
stimulus closely resemble those following penetration of uterine
epithelium by trophoblast cells (Fig. 12; 81-83, 63).
10: Experimental model to study endometrial tissue behaviour
to trophoblast penetration and to conceptus-derived
signals in mated Rhesus monkeys and that of deciduomatous
tissue of Rhesus monkey after the application of artificial
trauma to hormone-primed uterus.
for full view
11: The model as described in Figure 10 would allow
for a comparative understanding of tissue behaviour
in conception and artificial trauma- induced deciduorna
cycles of Rhesus monkeys.
for full view
12: Endometrial responses to deciduogenic stimulus (trauma)
given to hormone-primed Rhesus monkeys elicited endometrial
responses of edema, transformation of epithelial cells
to plaque acini, infiltration of natural killer (NK.)
type of endometrial granulated lymphocytes closely associated
with decidual cells and had close temporal and spatial
synchrony with the structural responses of primate endometrium
to implantation (63, 83).
In vitro model of blastocyst implantation
To investigate the cellular and molecular
biology of trophoblast-endometrial interaction, reproductive
biologists have been attempting to establish an in vitro model
of blastocyst implantation (84-88). It is now accepted that
experimental cell culture strategy must be developed (Fig.
13) in which endometrial epithelial cells and stromal fibroblasts
should manifest structural and functional polarization, express
typical phenotypic and differentiation responses under physiological
hormonal priming conditions, and such an experimental model
can then be used to study the three dimensional state of'
blastocyst adhesion and penetration of' epithelium in vitro
(89-94). Currently we are employing such an in vitro model
of' blastocyst implantation in our laboratory using three
dimensional cell culture system for rodent and primate blastocystendometrial
cell interactions (Figs. 14, 15). Three dimensional system
could thereby provi de an effective handle to study in timed
and sequential manner the physiology of blastocyst implantation
for full view
for full view
14: Structural responses of Rhesus monkey epithelial
cells grown in vitro for 10 davs on rat-tail
collagen in 10% fetal calf-serum (FCS) containing TCM199
(A). Mouse epithelial cells grown on basement membrane
extract in DMEM : F12 (I : 1) medium containing 10%
FCS showing ultrastructural features of cell polarity.
Bars : 20 mm (A), and 5
15: Lower power montage micrographs of Rhesus monkey
epithelial and stromal cells grown on rat-tail collagen
for 21 days initially in 10% FCS containing TCM199 for
7 days and then in serum-free TCM 159 supplemented with
estrogen, progesteronc and growth factors. Note the
significant degree of 'tissue-like' organisation of
luminal columnar epithelial cells and underlying stromal
cells in this heterotypic culture (A). Mouse zona-free
blastocyst showing apposition and initial stages of
adhesion to epithelial cells in heterotypic culture
(B). Bars ; 30 mm.
The studies reported from the authors’ laboratory were supported
by research grants funded by the Indian Council of Medical
Research, the Rockefeller Foundation, the WHO Special
Program of Research, Development and Research Training in
Human Reproduction and the Department of Science and Technology,
Government of India. We acknowledge the kind courtesy of
the Human Developmental Anatomy Center, Division of Collections
and Research, National Museum of Health and Medicine, Armed
Forces Institute of Pathology, Washington DC, USA, for examination
and reproduction of the Carnegei Collection of human implantation
stages 5a and 5b.
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