Experimental models for human implantation

JAYASREE SENGUPTA*¹ AND DEBABRATA GHOSH
Department  of Physiology,
All India Institute of Medical Sciences,
New  Delhi -    110 029
(Received on May 15, 2002)

 

Key words :blastocyst, implantation, placentation, endometrium, paracrine factors, decidualization, endocrine factors, receptivity, NK cells, primary cell culture

Introduction
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 and placentation
Inflammation paradigm of blastocyst implantation
In vitro model of blastocyst implantation
References

INTRODUCTION

Understanding 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.

Models in experimental biology

To 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).

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Fig. 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).

Experimental models for human implantation

In 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 discussion.
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Window of implantation

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 failure.

Embryo-endometrium 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).

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Fig. 2:  An experimental model to study the nature of embryo-endometrial interaction leading to the induction of receptive stage endometrium in the Rhesus monkey.

 

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Fig 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 (C).

The understanding 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).

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Fig 4. Model ofembryo-endometrial dialogue involving secretory signals from both compartments. B, blastocyst. Ep, epithelial cells. Bm, basement membrane. ECM, extra-cellular matrix. F, fibroblast. Gl, gland. V, blood vessel.

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Endocrinology of blastocyst implantation

The species-specificity 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).

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Fig. 5: An experimental model to study the requirement of ovarian estrogen and progesterone for blastocyst implantation in the primate using stage- 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.

 

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Fig. 6: Experimental models in the human and the Rhesus monkey to investigate the actions of luteal phase progesterone in blastocyst implantation.

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Paracrinology of blastocyst implantation

Mid-secretory 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

hormone 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).

A 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 a_vb₃ and a₄b₁ in human endometrium during the 'implantation  window' has been documented, and the lack a_vb₃ 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 PGF_2a (48), while in the Rhesus monkey, PGF₂ but not PGF_2a 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).

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Fig. 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).

Endocrine 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 Washington DC.  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). 

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Fig. 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 stage 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).

 

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Fig. 9: Experimental model for collection of timed gestational stage tissue from mated Rhesus monkeys for the study of stage-specific gene and protein 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).

Defective 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.
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Inflammation 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).

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Fig. 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.

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Fig. 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.

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Fig. 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 (95).

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Fig. 14. and Fig. 15.    Click for full view

Fig. 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 mm (B).   Fig. 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.

 ACKNOWLEDGEMENTS

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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REFERENCES 

1.       Hertig AT. Human Trophoblast: normal and abnormal. A plea for the study of the normal so as to understand the abnormal. Ward Burdick Award Address. Am J Clin Pathol 1967; 47: 249-268.

2.       Hertig AT. Gestational hyperplasia of endometrium. Lab invest 1964; 13: 1153-1191.

3.       Psychoyos A. Endocrine control of egg implantation. Greep RO, Astwood ED (eds): Handbook of Physiology, Washington DC. 1973; 187-215.

4.       Simon C, Landeras J, Zuzuarregui JL et al. Early pregnancy losses in in vitro fertilization and oocyte donation. Fertil Steril 1999; 72: 1061-1065.

5.       Bergh PA.Navot D. The impact of embryonic development and endometrial maturity bon the timing of implantation. Fertil Steril 1992; 58: 537-542.

6.       Ghosh D, Sengupta J. A comparative study of endometrial acid phosphatase activity in the mid- luteal phase of the menstrual cycle and in the peri- implantation stage in the rhesus monkey (Macaca mulatta). J Endocrinol 1988; 116: R7-R9.

7.       Ghosh D, Roy A, Sengupta J, Johannisson E.Morphological characteristics of preimplantation stage endometrium in the rhesus monkey. Hum Reprod 1993; 8:1579-1587.

8.       Ghosh D, Sengupta J. Patterns of estrogen and progesterone receptors in rhesus monkey endometrium during secretory phase of normal menstrual cycle and preimplantation stages of gestation. J Steroid Biochem 1988; 31: 223-229.

9.       Sengupta J, Sakhuja D, Paria BC, Ghosh D. Endometrial phOsphatases, P glucuronidase and cathepsin D during the menstrual cycle and pre-implantation stages of gestation in the rhesus monkey (Macaca mulatta.). Acta Endocrinol 1988; 118:142-146.

10.    Mazur MT, Duncan DA, Benjamin  Younger J. Endometrial biopsy in the cycle of conception: histologic and lectin histochemical evaluation. Fertil Steril 1989; 51: 764-769.

11.    Ghosh D, De P, Sengupta J. Luteal phase oestrogen is not essential for implantation and maintenance of pregnancy from surrogate embryo transfer in the rhesus monkey. Hum Reprod 1994; 9: 629-637.

12.    Ghosh D, Sengupta J. Endometrial receptivity for implantation. Another look at the issue of periimplantation oestrogen. Hum Reprod 1995; 10: 1-2.

13.    Edgar DH. Oestrogen and human implantation. Hum Reprod 1995; 10: 2-3.

14.    de Ziegler D. Hormonal control of endometrial receptivity. Hum Reprod 1995; 10: 4-7.

15.    Zeegers-Hoschild  F, Alteiri E.  Luteal  phase estrogen is not required for establishment of pregnancy in the human. J Assist Reprod Genet 1995; 12:224-228.

16.    de Ziegler D, Bergeron C, Cornel C et al. Effects of luteal estradiol on the secretory transformation of human endometrium and plasma gonadotrophins. J Clin Endocrinol Metab 1992; 74: 322-331.

17.    Gemzell-Daniellson K, Swahn ML, Svalander P, Bygdeman M. Early luteal phase treatment with mifepristone (RU486) for fertility regulation. Hum Reprod 1993; 8: 870-873.

18.    Ghosh D, Sengupta J. Anti-nidatory effect of a single, post-ovulatory  administration  of mifepristone (RU486) in the rhesus monkey. Hum Reprod 1993; 8:552-5S8.

19.    Roberts DK, Walker NJ, Lavia LA. Ultrastructural evidence of stromal/epithelial interactions in human endometrial cycle. Am J Obstet Gynecol 1988; 158:854-861.

20.    Cunha GR, Bigsby RM, Cooke PS et al. Stromal-epithelial interactions in adult organs. Cell Diff 1985; 17:137-148.

21.    Lessey BA, Killam AP, Metzger DA et al. Immunohistochemical analysis of human uterine oestrogen and progesterone receptors throughout the menstrual cycle. J Clin Endocrinol Metab 1988; 67:334-340.

22.    Ciocca DR, Asch RH, Adams DJ, McGuire WI. Evidence for modulation of a 24K protein in human endometrium. J Clin Endocrinol Metab 1983; 57: 496-499.

23.    Tabibzadeh S, Kong OF, Satyaswaroop PG, Babaknia A. Heat shock proteins in human endometrium throughout the menstrual cycle. Hum Reprod 1996; 11: 633-640.

24.    Koshiyama M, Konishi I, Nanbu K, Nanbu Y, Mandai M, Komatsu T, Yamamoto S, Mori T, Fujii S. Immunohistochemical localization of heat shock proteins HSP70 and HSP90 in the human endometrium; correlation with sex steroid receptors and Ki-67 antigen expression. J Clin Endocrinol Metab 1995; 80: 1106-1116.

25.    Manners CV. Endometrial assessment in a group of infertile women stimulated cycles for IVF: immunohistochemical findings. Hum Reprod 1990; 5:128-132.

26.    Okulicz WC, Ace Cl, Longcope C, Tast J. Analysis of differential gene regulation in adequate versus inadequate secretory-phase endometrial complementary  deoxyribonucleic acid populations from the rhesus monkey. Endocrinology 1996; 137: 4844-4850.

27.    Seppala M, Koistinen H, Koistinen R et al. Glycodelins as regulators of early events of reproduction. Clin Endocrinol 1997; 46: 381-386.

28.    Dalton CF, Laird SM, Serle E et al. The measurement of CA125 and placental protein 14 in uterine flushings in women with recurrent miscarriage: relation to endometrial morphology.Hum Reprod 1995; 10: 2680-2684.

29.    Delage G, Moreau JF, Taupin JL et al. In vitro endometrial secretion of human interleukin for DA cells/leukaemia inhibitory factor by explant cultures from fertile and infertile women.  Hum Reprod 1995; 10: 2483-2488.

30.    Ghosh D, Sengupta J. Recent developments in ndocrinology and paracrinology of blastocyst implantation in the primate. Hum Reprod Update1998; 4:153-168.

31.    Cowell TP. Implantation and development of mouse eggs transferred to the uteri of non-progestational mice. J Reprod Fertil 1969; 19: 239-245.

32.    Murphy CR. Junctional barrier complexes undergo major alterations during the plasma membrane transformation of uterine epithelial cells. Hum Reprod 2000; 15:182-188.

33.    Thie M, Fuchs P, Butz B et al. Adhesiveness of the apical surface of uterine epithelial cells : the role of junctional complex integrity. Eur J Cell Biol 1996; 70:221-232.

34.    Rusholati E. Integrins. J Clin Invest 1991; 87: 1-5.

35.    Tabibzadeh S. Patterns of expression of integrin molecules in human endometrium throughout the menstrual cycle. Hum Reprod 1992; 7: 876-882.

36.    Lessey BA, Damjanovich L, Coutfaris C et al. Integrin adhesion molecules in the human endometrium. Correlation with the normal and abnormal menstrual cycle. J Clin Invest 1992; 90: 188-195.

37.    Nikas G, Drakakis P, Loutradis D et al. Uterine pinopodes as markers of the 'nidation' window in cyclic women receiving exogenous oestradiol and progesterone. Hum Reprod 1995; 10: 1208-1213.

38.    Sengupta J, Ghosh D. Blastocyst-endometrium interaction at implantation in the rhesus monkey. J Reprod Immunol 2002; 53: 227-239.

39.    Ghosh  D, Sengupta J,  New perspectives  on immunorecognition of gametes and embryos. Molecular mechanism of embryo-endometrial adhesion in primates: a future task for implantation biologists. Mol Hum Reprod 1998; 4: 733-773.

40.    Graham RA, Li TC, Seif MW et al. The effects of antiprogesterone  RU486  mifepristone) on  an endometrial  secretory  glycan:  an immunocytochemical study. Fertil Steril 1991; 55:1132-1136.

41.    Aplin  JD,  Graham  RA,  Hey  NA.  Human endometrial MUCI carries keratan sulphate: characteristic glycoforms in the luminal epithelium at receptivity. Glycobiology 1998; 8: 269-276.

42.    Hoffman LH, Olsen GE, Carson DD, Chilton BS. Progesterone and implanting blastocyst regulate Muc 1 expression in rabbit uterine epithelium. Endocrinology 1998; 139: 266-271.

43.    Harvey MB, Leco KJ, Arcellana-Panlilio MY et al. Roles of growth factors during preimplantation development. Hum Reprod 1995: 10: 712-718.

44.    Sharkey AM, Dellow K, Blayney M et al. Stage-specific  expression  of cytokine  and receptor messenger ribonucleic acids in human preimplantation embryos. Biol Reprod 1995; 53: 955-962.

45.    Casalfi EM, Raga F, Polan ML. GnRH mRNA and protein expression in human preimplantation embryos. Mol Hum Reprod 1999; 5: 234-239.

46.    Fishel SB, Edwards RG, Evans PJ. Human chorionic gonadotrophin secreted by preimplantation embryos cultured in vitro. Science 1984; 223: 816-818.

47.    Edgar DH, James JB, Mills JA. Steroid secretion by human early embryos in culture. Hum Reprod 1993; 8:277-278.

48.    Holmes PV, Sjogren A, Hamberger L. Prostaglandin E2 released by preimplantation human conceptus. J Reprod Immunol 1989; 17: 79-86.

49.    Ghosh D, Sengupta J. Prostaglandins and blastocyst implantation in primates. Sengupta J, Ghosh D (eds):  Cellular and Molecular Signaling in Reproduction. New Delhi : New Age International, 1995; 145-162.

50.    Ghosh D, Sengupta J. Stage-specific secretion of de novo synthesized proteins by normal and abnormal rhesus monkey embryos in vitro. Indian J Physiol Pharmacol (Submitted).

51.    Singer-Sam J, Grant M, LeBon JM et al. Use of HpaII-polymerase chain reaction assay to study DNA methylation in the Pgk-l CpG island of mouse embryos at the time of X-inactivation. Mol Cell Biol 1990; 10:4987-4989.

52.    Poirier E, Timmins PM. Chan CT et al. Expression of L14 lectin during mouse embryogenesis suggests multiple roles during pre- and spot-implantation development. Development 1992; 115: 143-155.

53.    Antczak M, Van Blerkorn J. Oocyte influences on early development : leptin and STAT3 are polarized in mouse and human oocytes and differentially distributed within the cell of the preimplantation embryo. Mol Hum Reprod 1999; 3: 1067-1086.

54.    Plachot M, Belaisch-Allart J, Mayenga JM et al. Blastocyst stage transfer: the real benefits compared with early embryo transfer. Hum Reprod 2000; 15(Suppl 6): 24-30.

55.    Ghosh D, Sengupta J, Hendrickx AG. Effect of single dose, early luteal phase administration of mifepristone (RU486) on implantation stage endometrium in the rhesus monkey. Hum Reprod 1996; 11: 2026-2035.

56.    Ghosh D, Lalitkumar PGL, Sengupta J. Early luteal phase administration of mifepristone inhibits preimplantation embryo development and viability in the rhesus monkey. Hum Reprod 1997; 12: 575- 582.

57.    Ghosh D, Lalitkumar PGL, Wong V et al. Preimplantation embryo morphology following early luteal phase anti-nidatory treatment with mifepristone (RU486) in the rhesus monkey. Hum Reprod 2001; 15:180-188.

58.    Rubio C, Simon C, Mercader A et al. Clinical experience employing co-culture of human embryos with autologous human endometrial epithelial cells. Hum Reprod 2000; 6: 31-38.

59.    Hertig AT, Rock J. Two human ova of the previllous stage having an ovulation age of about eleven and twelve days respectively. Contrib Embryology 1941; 29:127-156.

60.    Heuser CH, Streeter GL. Development of the macaque embryo. Contrib Embryol 1941; 29: 15-56.

61.    Hendrickx AG. Embryology of the baboon. Chicago: The University of Chicago Press, 1971.

62.    Enders AC, Schlafke S. Differentiation of blastocyst of the rhesus monkey. Am J Anat 1981; 162: 1-21.

63.    Enders AC. Current topic : Structural responses of the primate endometrium to implantation. Placenta1991; 12:309-325.

64.    Enders AC, Welsh AO, Schlafke S. Implantation in the rhesus monkey: endometrial responses. Am J Anat 1985; 173:147-169.

65.    Glasser SR, McCormick S. Functional development of rat trophoblast and decidual cells during establishment of the hemochorial placenta.Kaye AM, Kaye M (eds): The Development of Responsiveness to Steroid Hormones. Pergamon Press, 1980.

66.    Ghosh D, Dhara S, Kumar A, Sengupta J. Immunohistochemical localization of receptors for progesterone and oestradiol-17bp in the implantation site of the rhesus monkey. Hum Reprod 1999; 14:505-514.

67.    Salmi A, Anmala M, Rutanen EM. Proto-oncogenes c-jun and c-fos are down-regulated in human endometrium during pregnancy: relationship to oestrogen receptor status. Mol Hum Reprod 1996: 2:979-984.

68.    Stewart CEH, Rotwein P. Insulin like growth factor II is an autocrine survival factor for differentiating myoblasts. J Biol Chem 1996; 271: 11330-11336.

69.    Dhara S, Lalitkumar PGL, Sengupta J, Ghosh D.Immunohistochemical localization of insulin-like growth factors I and II at the primary implantation site in the Rhesus monkey. Mol Hum Reprod 2001; 7:365-371.

70.    Irving JA, Lala PK. Functional role of cell surface integrin on human trophoblast cell migration: regulation by TGFb, IGFII and IGFBP1. Exp Cell Res 1995; 217: 419-427.

71.    Irwin JC, de las Fuentas L, Duspin BA, Guidice LC. Insulin like growth factor regulation of human endometrial .stromal cell function: coordinate effects on insulin-like growth factor binding protein I, cell proliferation and prolactin secretion. Regul Peptides 1993; 48:165-177.

72.    Jones Jl, GockermanA, Busby Jr WH et al. Insulin like growth factor binding protein I stimulates cell migration and binds to a5pl integrin by means of its Arg-Gly-Asp sequence. Proc Natl Acad Sci USA 1993; 90:10553-10557.

73.    Ghosh D, Sharkey AM, Charnock-Jones DS et al. Expression of vascular endothelial growth factor (VEGF) and placental growth factor (PIGF) in conceptus and endometrium during implantation in the rhesus monkey. Mol Hum Reprod 2000; 6:935-941.

74.    Khaliq A, Dunk C, Jiang J, Shams M et al. Hypoxia down-regulates placenta  growth factor, whereas fetal growth restriction up-regulates placenta growth factor expression: molecular evidence for "placental hyperoxia" in intrauterine growth restriction. Lab Invest 1999; 7: 151-170.

75.    Genbacev 0, Joslin R, Damsky CH et al. Hypoxia alters early gestation human cytotrophoblast differentiation/invasion in vitro and models the placental defects that occur in pre-eclampsia. J Clin invest 1996; 97: 540-550.

76.    Papapetropoulos A, Garcia-Cardena G, Madri JA, Sessa W. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J Clin Invest 1997; 100:3131-3139.

77.    Dunk C, Shams M, Nijjar S et al. Angiopoietin-1 and angiopoietin-2 activate trophoblast Tie-2 to promote growth and migration during placental development. Am J Pathol 2000; 156: 2185-2199.

78.    King A. Uterine leucocytes and decidualization. Hum Reprod 2000; 6: 28-36.

79.    Loke YW, King A. Immunological aspects of human implantation. J Reprod Fertil (Suppl) 2000; 55: 83-90.

80.    Finn CA. Implantation, menstruation and inflammation. Biol Rev Camb Philos Soc 1986; 61: 313-328.

81.    Ghosh D, Sengupta J. Endometrial responses to a deciduogenic stimulus in ovariectomized rhesus monkeys treated with oestrogen and progesterone.J Endocrinol 1989; 120: 51-58.

82.    Sengupta J, Given RL, Talwar D, Ghosh D. Endometrial response to deciduogenic stimulus in ovariectomized rhesus monkeys treated with oestrogen and progesterone: an ultrastructural study. J Endocrinol 1990; 124: 53-57.

83.    Sengupta J, De P, Ohosh D. Implantation of the primate embryo. Curr Sci 1995; 68: 363-374.

84.     Glenister  TW.  Observations  on  mammalian blastocysts implanting inorgan culture. Enders AC (ed):  Delayed  Implantation.  Chicago:  University of Chicago Press, 1963.

85.    Grant PS, Ljungkvist I, Nilsson O. The hormonal control and morphology of blastocyst invasion in the mouse uterus in vitro. J Embryol Exp Morphol 1975; 34:200-210.

86.    Sengupta J, Given RL, Carey JB et al. Primary culture of mouse endometrium on floating collagen gels: a potential in vitro model for implantation. Ann NY Acad Sci 1986; 476: 75-94.

87.    Glasser SR, Mulholland J. In vitro models of implantation. Strauss JF, Lyttle CR (eds) : Uterine and Embryonic Factors in Early Pregnancy. New York: Plenum Press, 1991; 33-50.

88.    Bentin-Ley U, Horn T, Sjogren A et al. Ultrastructure of human blastocyst-endometrial interactions in vitro. J Reprod Fertil 2000; 120: 337-350.

89.    Ghosh D, Sengupta J. Morphological characteristics of human endometrial epithelial cells cultured on rat tail collagen matrix. Hum Reprod 1995; 10: 785-790.

90.    Ghosh D, Danielson KG, Alstone JT, Heyner S.Functional differentiation of mouse uterine epithelial cells grown on collagen gels or reconstituted basement membranes. In Vitro Celt Dev Biol 1991; 27A: 713-719.

91.    Glasser SR, Julian J, Decker GL et al. Development of functional polarity in primary cultures of immature rat uterine epithelial cells. J Cell Biol 1988; 107:2409-2923.

92.    92.  Julian JA, Carson DD, Glasser SR. Polarized rat uterine epithelium in vitro. Responses in defined medium. Endocrinology 1992; 130: 68-78.

93.    Ghosh D. Primary culture  of mouse  uterine epithelial cells:  Endocrine  and  paracrineregulation. Ghosh D, Sengupta J (eds) : Frontiers in Reproductive Physiology. New Delhi: Wiley Eastern, 1992; 95-103.

94.    Venkatraman L, Upadhyay S, Lalitkumar PGL, Sengupta J, Ghosh D. Morphological and functional characteristics of rabbit uterine epithelial cells grown on free floating collagen gel. Indian J Physiol Pharmacol 2001; 45: 161-171.

95.    Sharpe-Timms KL, Glasser SR. Models for the study of uterine receptivity for blastocyst implantation.  Sem Reprod Endocrinol 1999; 17:107-115.