Francisco Domínguez

Controlled Ovarian Stimulation Induces a Functional Genomic Delay of the Endometrium with Potential Clinical Implications

CONTEXT Controlled ovarian stimulation induces morphological, biochemical, and functional genomic modifications of the human endometrium during the window of implantation. OBJECTIVE Our objective was to compare the gene expression profile of the human endometrium in natural vs. controlled ovarian stimulation cycles throughout the early-mid secretory transition using microarray technology. METHOD Microarray data from 49 endometrial biopsies obtained from LH+1 to LH+9 (n=25) in natural cycles and from human chorionic gonadotropin (hCG) +1 to hCG+9 in controlled ovarian stimulation cycles (n=24) were analyzed using different methods, such as clustering, profiling of biological processes, and selection of differentially expressed genes, as implemented in Gene Expression Pattern Analysis Suite and Babelomics programs. RESULTS Endometria from natural cycles followed different genomic patterns compared with controlled ovarian stimulation cycles in the transition from the pre-receptive (days LH/hCG+1 until LH/hCG+5) to the receptive phase (day LH+7/hCG+7). Specifically, we have demonstrated the existence of a 2-d delay in the activation/repression of two clusters composed by 218 and 133 genes, respectively, on day hCG+7 vs. LH+7. Many of these delayed genes belong to the class window of implantation genes affecting basic biological processes in the receptive endometrium. CONCLUSIONS These results demonstrate that gene expression profiling of the endometrium is different between natural and controlled ovarian stimulation cycles in the receptive phase. Identification of these differentially regulated genes can be used to understand the different developmental profiles of receptive endometrium during controlled ovarian stimulation and to search for the best controlled ovarian stimulation treatment in terms of minimal endometrial impact.

Embryonic implantation and leukocyte transendothelial migration: different processes with similar players?

A clear parallelism between the different steps in human embryo‐endometrial apposition/adhesion/invasion and leukocyte‐endothelium rolling/adhesion/extravasation can be established. During human implantation and leukocyte trafficking, a first wave of soluble mediators regulates the expression and functional activity of adhesion molecules such as L‐selectin and integrins, which mediate both processes. Apical surfaces of human endometrial epithelium and endothelium are key elements for the initiation of molecular interactions to capture the blastocyst or leukocyte, respectively. Subsequently, the blastocyst and the leukocyte migrate through the epithelium and endothelium toward their final destination, the endometrial stroma, to initiate placentation or the inflammatory foci as part of the immune response. Similarities between the intermediate molecular mechanisms of these two physiologically unrelated processes are discussed. Dominguez, F., Yáñez‐Mó, M., Sanchez‐Madrid, F., Simón, C. Embryonic implantation and leukocyte trans‐endothelial migration: different processes with similar players? FASEB J. 19, 1056–1060 (2005)

The role of the leptin in reproduction.

Purpose of review Since its discovery in 1994, leptin has appeared to be a pleiotrophic hormone, governing energy homeostasis and affecting many tissues in the body. Numerous pieces of evidence have accumulated showing that leptin potentially plays an important role in the control of the reproductive function. Recent findings This review presents the major concepts for the role of leptin in the modulation of reproductive function. As a marker of the nutritional status, leptin affects the hypothalamo–pituitary–gonadal axis, regulating gonadotrophin-releasing hormone and luteinising hormone secretion, and appears to be a permissive factor in the onset of the puberty. This protein and its receptor have been found in the reproductive tissues, indicating that this system could be also implicated in several processes such as embryo development, implantation and pregnancy. Moreover, disorders of the leptin system have been related to some reproductive pathologies such as pre-eclampsia and polycystic ovary syndrome. However, controversy surrounds several aspects of the action of leptin in reproduction that require a deeper investigation of this system. Summary Results to date suggest that this system could be implicated in important reproductive processes such as embryonic development and implantation. Moreover, understanding the role of leptin might be useful for new treatments in reproductive pathologies.

Comparative protein-profile analysis of implanted versus non-implanted human blastocysts

BACKGROUND New approaches for non-invasive embryo-quality assessment are among the major goals in Reproductive Medicine. We hypothesize that the detection of changes in the protein profile of the culture media in which blastocysts are cultured could be a potential indicator of the viability of the embryo and, thus, a useful tool for selecting the more appropriate blastocysts to be transferred. METHODS Using protein-array technology, we analysed the protein profile corresponding to 24 h conditioned media of blastocysts that implanted versus those that did not implant. A statistical approach was followed to compare each of these media versus a medium in the absence of blastocysts (control medium). In addition, a gene ontology functional analysis-including those proteins showing a statistical difference among conditions-was performed, and a network with the predicted functional partners and corresponding relationships was obtained. RESULTS The soluble TNF receptor 1 and IL-10 increased significantly and MSP-alpha, SCF, CXCL13, TRAILR3 and MIP-1beta decreased significantly when the protein profile of the blastocyst culture medium was compared with the control medium. CXCL13 (BLC) and granulocyte-macrophage colony-stimulating factor was also decreased significantly in the implanted blastocyst media compared with that in media from the non-implanted counterparts with a similar morphology. None of the proteins included in the array was increased significantly in the implanted blastocyst-conditioned media. CONCLUSIONS The differences identified in the protein profile of the culture media in the presence of implanted versus non-implanted blastocysts can be considered a new non-invasive approach in the search for new tools to diagnose blastocyst viability.

Proteomic analysis of the human receptive versus non-receptive endometrium using differential in-gel electrophoresis and MALDI-MS unveils stathmin 1 and annexin A2 as differentially regulated

BACKGROUND The transcriptome of the endometrium throughout the menstrual cycle has been described in recent years. However, the proteomic of the window of implantation remains unknown. The aim of this study was to compare the proteome of the human endometrium in the pre-receptive phase versus the receptive phase by identifying and quantifying the proteins differentially expressed using differential in-gel electrophoresis (DIGE) and mass spectometry (MS). METHODS Endometrial biopsies were collected at days 2 (pre-receptive) and 7 (receptive) after the urinary luteal hormone surge in the same menstrual cycle from eight fertile women (corresponding to days 16 and 21 of the menstrual cycle). Proteins were extracted and labeled with CyDye DIGE fluorofores and separated using two-dimensional gel electrophoresis. RESULTS Image analysis using the DeCyder software followed by protein identification by matrix-assisted laser desorption/ionization-MS and database searching revealed 32 differentially expressed proteins, although only annexin A2 and stathmin 1 were consistently regulated in the two experiments performed. Validation and localization of annexin A2 and stathmin 1 were performed by western blot and immunohistochemistry. Annexin A2 and stathmin 1 were investigated using an endometrial refractoriness model. The results highlight the key potential of these proteins as possible targets for human endometrial receptivity and interception. CONCLUSION This study shows that the human endometrium has a differential proteomic repertoire during the window of implantation. Consequently, we identified annexin A2 and stathmin 1 as differentially expressed molecules in the receptive endometrium.

Determinants of Endometrial Receptivity

Abstract: Understanding the molecular changes that occur during the window of implantation is fundamental to our knowledge of human reproduction. Lately, the development of microarray technology has allowed this process to be studied from a global molecular perspective. In the last 2 years, researchers have focused their efforts on throwing light on the gene expression profile of the receptive endometrium. The genes hold the key to the development of the endometrium at any stage, and we have focused our work on the window of implantation. The four most recently published works in this field have revealed a long list of genes that are up‐ or downregulated at the time of implantation. Although these studies have been conducted using varying approaches, collectively these studies identify new candidate markers that can be used to accurately diagnose the receptive state of the endometrium. The next step is to perform functional analysis for confirming the importance of these genes. In this article, we gather together these recent findings to provide an overview of the current knowledge regarding the genetic functioning of human endometrial receptivity and related processes.

Embryologic outcome and secretome profile of implanted blastocysts obtained after coculture in human endometrial epithelial cells versus the sequential system.

OBJECTIVE To compare embryologic and clinical outcomes in terms of preimplantation development, implantation, pregnancy rates, and secretome profile of implanted blastocysts from the preimplantation genetic diagnosis program grown in sequential versus endometrial epithelial cell (EEC) coculture system. DESIGN Retrospective clinical study and prospective experimental study. SETTING In vitro fertilization clinical unit and university research laboratory. INTERVENTION(S) Blastomere biopsy, embryo culture, blastocyst transfer, and protein analysis of the media conditioned from implanted embryos obtained from coculture and sequential systems. MAIN OUTCOME MEASURE(S) Clinical study: blastocyst, implantation, and gestation rates in own and donated oocytes. Experimental study: differential protein analysis of implanted embryos grown in coculture system versus sequential system. RESULT(S) Of the 12,377 embryos analyzed, the blastocyst rates were 56.0% versus 45.9% in the coculture versus the sequential system, respectively, with own oocytes. With ovum donation, the rates were 70.5% versus 56.4%, respectively. Reproductive outcomes in terms of pregnancy rates (39.1% vs. 27.5%) and implantation rates (33.3% vs. 20.9%,) were statistically higher in EEC coculture versus sequential media. Furthermore, the protein profile of the EEC coculture versus the sequential system was obtained. Interleukin-6 (IL-6) was the most secreted protein by the EEC culture. Further ELISA experiments showed that the IL-6 present in the sequential medium diminished in implanted blastocysts. CONCLUSION(S) The coculture system favors blastocyst development and implantation rates, given the contribution of the factors secreted by endometrial epithelial cells, such as IL-6.

CXCL10 and IL-6 induce chemotaxis in human trophoblast cell lines

The investigation of trophoblast chemoattractive molecules in humans is of high interest for the reproductive field. Current evidence in ruminants demonstrates that CXCL10, formerly the interferon-gamma-inducible protein 10 (IP-10), is a potent chemotactic molecule implicated in the migration of trophoblast cells during early gestation. The aim of this work was to explore the existence of CXCL10/CXCR3 in the human model. Furthermore, chemotaxis assays were performed to demonstrate CXCL10 chemotactic activity in the human trophoblast cell lines JEG-3 and AC-1M88. Surprisingly, the conditioned media from epithelial endometrial cells (EEC) induced the highest trophoblast migration rate. Cytokine and chemokine membrane protein arrays were used to identify the secreted protein profile of EEC-conditioned media, and IL-6 was found to be the most abundant and CXCL13 the second most abundant molecule. Using a chemotaxis assay on AC-IM88, IL-6 antibody blocked the effect of EEC, indicating IL-6 to be an effective chemoattractive factor for trophoblast cells in the human model.

Embryo Effects in Human Implantation

Embryonic implantation requires coordinated development of the blastocyst and the maternal endometrium. Considerable advances have been made in the understanding of the cell biology of human embryo and maternal endometrium as separate entities. Nevertheless, communication between them and their reciprocal effects on each other constitute an exciting and as‐yet unsolved problem in reproductive medicine. Cross‐talk among the embryo, endometrium, and the corpus luteum are known to occur in ruminants and primates; more specifically, endometrial‐embryonic interactions have been reported in rodents and primates. Here, we present updated information in humans on the embryonic regulation of endometrial epithelial molecules such as chemokines, adhesion molecules, antiadhesion molecules, and leptin during the apposition and adhesion phases of human implantation. Also the embryonic induction of apoptosis in endometrial epithelial cells as a mechanism for crossing the epithelial barrier will be described.

The role of estrogen in uterine receptivity and blastocyst implantation

Abstract The endometrium is a specialized, hormonally regulated organ that is non adhesive for embryos throughout most of the reproductive cycle in mammals. Thus, the window of implantation is a self-limited period in which the endometrial epithelium (EE) acquires a functional and transient ovarian steroid-dependent status. The luminal EE initiates the adhesion of the developing blastocyst during this period, owing mainly to the presence of progesterone (P) after appropriate 17β-estradiol (E 2 ) priming in humans or because of the addition of E 2 after appropriate P priming in rodents. Wen-ge et al. have now demonstrated in mice that low levels of exogenous E 2 can maintain the window of receptivity for an extended period of time, whereas high doses of E 2 can rapidly initiate a refractory state. In summary, levels of E 2 within a very narrow range determine the duration of the window of implantation in uterine receptivity in mice. These outstanding results demonstrate the possibility of manipulating the receptivity window with the use of different doses of E 2 .