A systems biology approach towards understanding the process of blastocyst implantation.

Time-synchronous development of endometrium and embryo under adequate progesterone dominance is considered integral to the process of blastocyst implantation in the human. It now appears that hypothesisdriven and candidate-based deductive approach fails to explain this complex control process. We propose a systems biology approach to elucidate the control process underlying the physiological basis of successful interfacing between embryo and endometrium towards blastocyst implantation. Elucidation of the time course pattern of transcriptomics involved in the process of blastocyst implantation in mid-luteal phase endometrium with and without progesterone dominance, as well as, with and without viable embryo shall elaborate upon the polygenic and multifactorial nature of the process of blastocyst implantation. Accordingly, a large scale homeodynamic model of hierarchical arrangement of functional networks of regulatory genomic expressional elements at the level of endometrial receptivity shall emerge. It is anticipated that such a systems biology approach shall provide an integrated picture of the process and shall also open up novel areas of basic, strategic and translational research in the biology of blastocyst implantation.

Effect of human chorionic gonadotropin (hCG) on expression of vascular endothelial growth factor a (VEGF-a) in human mid-secretory endometrial cells in three-dimensional primary culture.

Several lines of evidence suggest that human uterine endometrial cells can bind human chorionic gonadotropin (hCG) which, in turn, influences the physiology of implantation stage endometrium. Vascular endothelial growth factor (VEGF) appears to be a candidate mediator in this process. However, our knowledge about hCG action on VEGF in human endometrial cells is very thin. In the present study, we have examined microscopically hCG binding to dissociated human endometrial cells collected from mid-luteal phase and maintained in three-dimensional primary co-culture on rat-tail collagen type I biomatrix and examined the effect of different concentrations (0, 1, 10, 100 and 1000 IU/ML) of hCG on VEGF expression and secretion by endometrial cells maintained in the above system. We report that both cytokeratin positive epithelial cells as well as vimetin positive stromal cells from human mid luteal phase endometrium could bind hCG and that their number increased (P < 0.01) steadily with time. Administration of hCG enhanced (P < 0.05) immunoreactive VEGF protein expression in dose dependent manner in endometrial cells retrieved from mid-luteal phase of cycle, and co-cultured in a three-dimensional cell culture system, but with no marked change in VEGF secretion. Collectively, it appears that hCG influences VEGF protein synthesis in human midluteal phase endometrial cells, but has little effect on post-translational regulation and secretion. From physiological homeostasis point of view, it is likely that synthesis and secretion of VEGF exhibits a modular and factorial regulation to achieve a fine tuning of this potent vasotropic agent in receptive stage endometrium.

Molecular correlates of syncytialization in muscle and placenta.

Syncytialization is one of the most fundamental processes in life. It is observed during development of muscle and osteoclast, and syncytiotrophoblast formation in placental villi. Syncytialization involves recognition, migration, adhesion and finally cell fusion between two interacting cells. It is an energy-dependent process which is essentially restricted to a small portion of interacting cellular membranes. Such regions of membranes may differ from other regions of cell surface in terms of physico-chemistry and expression of specific protein biomolecules resulting in restriction of this process to cells of specific competence. Despite the fact that membrane biologists have given significant quanta of efforts to understand the basic principle underlying this fundamental process of life, further large scale initiatives have to be undertaken to dissect the underlying molecular correlates central to this event.

In vitro action of leukemia inhibitory factor (LIF) on mid-secretory stage endometrial stromal cells collected from hormone-simulated, ovariectomized monkey and maintained in three-dimensional primary culture.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that is known to play an important role in blastocyst implantation. The putative action of LIF in the regulation of uterine function has been examined using mid-secretory stage monkey endometrial stromal cells cultured on rat-tail collagen type I and treated with recombinant human LIF (rhLIF) or immunoneutralized LIF (in LIF) under serum-free condition. Long-term ovariectomized rhesus monkeys (n=8) underwent simulation of their menstrual cycles with steroid hormones and endometrial tissue samples were collected on cycle day 18; stromal cells were isolated and grown in primary culture on three-dimensional collagen matrix. Significant decline in cellular protein synthesis (P < 0.01) and cell proliferation index (P < 0.05) was observed in cells with increasing doses (0-1000 ng/ml) of rhLIF under serum-free in vitro condition. JAK1 expression in cultured cells increased (P < 0.01) in response to rhLIF as revealed from Western blot and confocal laser scanning microscopic examination, STAT1 and STAT2 expressions were unchanged, while pSTAT3 expression increased (P < 0.01) with increased concentration of rhLIF in culture medium. Autophosphorylation of JAK1 in endometrial stromal cells showed no change with increasing concentration (0.01 to 100 ng/ml) of rhLIF in vitro, but significant (P < 0.05) increase was observed with the time of exposure to rhLIF. Immunoneutralization of LIF or no addition of rhLIF to cultured cells led to significant (P < 0.01) increase in stromal cell proliferation index and significant (P < 0.01) decrease in the level of JAK1 and its autophosphorylation as compared to cells exposed to rhLIF alone. From the present set of experiments we conclude that rhLIF affects the physiological behaviour of monkey mid secretory stage endometrial stromal cells in vitro via the JAK-STAT signaling pathway.

Accessing modern contraceptive technologies in India.

The 'unmet' need for reproductive health care in India requires a wide demand net. Besides 'supply' of oral contraceptives, condoms and IUDs, the 'non-supply' approach for increasing contraceptive prevalence is recommended through education about the variety of safe contraceptives and awareness in the use of traditional methods. Expansion of education is partly responsible for the recent decline in fertility in India, however, gender inequality still prevails with poor sex ratio in urban regions, and deficit of around 35 million girls and women from higher mortality compared to males till the age of 30. The community can become a key force in knowledge sharing for greater contraceptive prevalence and also to support its service sector. Practice of.women's reproductive health involves complex socio-cultural-biological interaction in accessing modern contraceptive technologies. A number of interactive factors have been suggested to operate at different hierarchical levels to provide homeodynamic stability to the central event of reproductive health care under a central controller action, the government. While guidelines of the World Health Organization for quality care in family planning should be implemented in rural and urban sectors, efforts should also be made to translate recent advances in contraceptive technology from laboratory to service sector for improving women's reproductive health.

Effect of tumour necrosis factor-alpha (TNF-alpha) on protein synthesis by mouse preimplantation stage embryos in vitro.

Successful blastocyst implantation depends upon the synchronous dialogue between age- and stage-matched embryo and adequately primed maternal endometrium. Endometrial signals present in the uterine lumen influence the growth and the viability of preimplantation stage embryo. Thus, uterine secretion of embryotoxic cytokines may affect the preimplantation stage embryo. Our previous study in the rhesus monkey has indicated that tumor necrosis factor-alpha (TNF-alpha) is one such candidate present in the uterine lumen, which may act as an embryotoxic agent. In the present study, the effect of TNF-alpha on de novo protein synthesis by mouse morulae (n = 100) and blastocysts (n = 100) in vitro was investigated by 2D-polyacrylamide gel electrophoresis. A total of 35 and 40 protein spots were detected in lysates of control morulae and blastocysts, respectively. Exposure of embryos to TNF-alpha (50 ng/ml) reduced the number of protein spots to 15 and 17 compared to that of control morulae and blastocysts. Seven spots in morula and 13 protein spots in blastocyst flourograms showed quantitative changes in their expressions with exposure to TNF-alpha. Morulae and blastocysts exposed to TNF-alpha expressed 8 and 17 protein spots, respectively, that were not seen in control embryos. It appears from the present study that exposure of preimplantation stage embryos to TNF-alpha affects their protein synthesis both quantitatively and qualitatively.

Effect of (Ala8,13,18)-magainin II amide on human trophoblast cells in vitro.

Magainins are cationic peptides with anti-bacterial and anti-tumor properties. The anti-nidatory function of a synthetic analogue of magainin, (Ala8,13,18)-magainin II amide, has earlier been reported, and it has been indicated that placental trophoblast cells could be a target of magainin resulting in its contragestational action. The aim of the present study was to examine the effect of (Ala8,13,18)-magainin II amide (100 ng/ml and 1000 ng/ml) on attachment efficiency, viability, differentiation in terms of hCG secretion and invasive function of isolated first trimester, human placental trophoblast cells grown on rat-tail collagen type-I matrix in primary cell culture. In the present experimental model, magainin was not found to affect human trophoblast cell functions in vitro.

Immunohistochemical localization of macrophage CD68+, HLA-DR+, L1+ and CD44+ subsets in uterine endometrium during different phases of menstrual cycle.

Different tissue macrophage subsets were immunohistochemically examined in normal endometrial samples collected from proliferative (n=4), peri-ovulatory (n=6) and secretory (n=8) phases of menstrual cycles in women. The different macrophage subsets, namely CD68 (pan macrophage marker), CD44 (transmembrane adhesion molecule), HLA-DR (transmembrane heterodimeric protein involved in antigen presentation) and L1 (calprotectin)-positive cells, as well as, CD45 (common leucocytic antigen)-positive cells were examined on the basis of immunohistochemical staining, and areas of immunoprecipitation were analyzed morphometrically using computer-assisted video imaging system. The stage-specific distribution of receptors for estrogen (ER) and progesterone (PR) in endometrial cells were examined and morphometrically analyzed. There was an increase in the number of CD45+ cells (P < 0.01) and CD68+ cells (P < 0.05) in secretory phase endometrium compared with proliferative and peri-ovulatory phases. There was no remarkable cycle dependent pattern in HLA-DR+ and L1+ cells. However, there was an increase in CD44 immunopositive area in peri-ovulatory (P < 0.05) and in secretory (P < 0.01) phases of endometrium compared with proliferative phase endometrium. A higher (P < 0.01) degree of immunopositivity for ER was observed during peri-ovulatory phase, and for PR, during peri-ovulatory (P < 0.05) and secretory (P < 0.01) phases compared with proliferative phase of cycle. Positive correlations between areas occupied by (i) CD68+ cells and PR (P < 0.01), (ii) HLA-DR+ and L1+ cells (P < 0.05), (iii) CD45+ and CD68+ cells (P < 0.01), (iv) CD45+ and L1+ cells (P < 0.05), and (v) PR and L1+ cells (P < 0.05) were obtained. It appears that the recruitment of different macrophage subsets in human endometrium involves a complex set of endocrine and paracrine factors.

Endocrine and paracrine correlates of endometrial receptivity to blastocyst implantation in the human.

Synchronous attainment of maternal endometrial receptivity allows implantation-stage adhesive blastocyst to undertake apposition, attachment and invasion. In the present essay, we propose a model according to which luteal phase progesterone induces a basic drive in endometrium toward receptivity and as a result, adequately primed endometrium differentiates through certain steps in a fixed action pattern. The implantation-stage embryo senses such endometrial responsiveness circumstantially by the factors secreted by maternal endometrium and undertakes differentiation to implant by secreting factors which act on maternal endometrial cells to further potentiate them to implantation stage-specific changes. Such a dynamic temporo-spatial manner of interaction involving a set of specific factors acting synchronously leads to the activation of innate releasing process in both compartments towards embryo attachment followed by successful intrusion and controlled invasion of trophoblast cells into maternal endometrium. In the present review we discuss the potential role of various endocrine and paracrine factors in the process of blastocyst implantation in the human.

Experimental models for human implantation.

An understanding of the cellular and molecular basis of blastocyst implantation in the human remains as yet a black box, however, a few experimental models using human and non-human primate species have addressed this issue. This review attempts to highlight, based on experimental evidence, the paradigm shifts in our understanding of the endocrine basis of embryo implantation, and the nature of dialogue between a growing, viable conceptus and maternal endometrial cells in the establishment of 'receptivity' for blastocyst implantation. It is being proposed that an existing inflammation paradigm of blastocyst implantation could be tested using an experimental model to compare tissue behaviour of conceptus associated endometrial cells with that occurring after induction of deciduoma in hormone-primed uterus. We anticipate that an in vitro model of blastocyst implantation using the experimental models of homotypic and heterotypic cultures of uterine epithelial and stromal fibroblast cells expressing structural and functional phenotypic responses as observed in situ may provide us with necessary clues about the temporal and spatial nature of cellular and molecular functions involving various endocrine and paracrine factors at implantation.

Effect of vaginally administered fumagillin on immunohistochemical distribution of leukemia inhibitory factor, interleukin 6, transforming growth factor beta and vascular endothelial growth factor in implantation stage endometrium of the rhesus monkey.

Intravaginal administration of an anti-angiogenic agent, fumagillin, during blastocyst implantation inhibits pregnancy establishment in a dose-related manner in the rhesus monkey. In the present study, mated female rhesus monkeys were vaginally inserted with tampons containing vehicle (group 1; n = 5) and test agent (fumagillin, 4 mg/animal; group 2; n = 6) on cycle day 20, and endometrial tissue samples were collected on cycle day 24 from all monkeys and processed for histological examination and immunohistochemical localization for LIF, IL-6, TGF-beta and VEGF. Concentrations of estradiol-17 beta, progesterone and chorionic gonadotrophin in peripheral circulation were determined. From the serum profiles of the hormones, 2 monkeys in group 1, and 1 monkey in group 2 appeared pregnant. However, endometrial morphology revealed histological evidence of pregnancy in 3 out of 6 fumagillin-treated animals. Histometric analysis of immunohistochemical staining in epithelial, stromal and vascular compartments revealed that per cent areas occupied by immunoprecipitate for the cytokines studies did not change in epithelial and stromal compartments, except that for TGF-beta which was higher (P < 0.05) in epithelial compartment in group 2. No change was observed in immunoprecipitation areas for IL-6 in epithelial, stromal and vascular compartments. On the other hand, changes (P < 0.05) for LIF, TGF-beta and VEGF were evident in the vascular compartment. It is possible that disparate responses observed in glandular, stromal and vascular compartments in implantation stage endometrium following fumagillin treatment actually caused from associated decline in progesterone concentration in peripheral circulation. It is also possible that fumagillin, an angiostatic agent, affects the synthesis and secretion of cytokines primarily in the vascular compartment of implantation stage endometrium, and thereby manifests differential responses in epithelial, stromal and vascular compartments.