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.

The Effect of Copper on Endometrial Receptivity and Induction of Apoptosis on Decidualized Human Endometrial Stromal Cells.

Intrauterine devices (IUDs) have been widely used to prevent pregnancies with great efficacy during decades. It has been demonstrated that IUD alters the endometrial gene expression, but there is no scientific data about how copper, a metal commonly used in these devices, by itself, is able to influence the processes of endometrial receptivity and apoptosis in decidualized human endometrial stromal cells. Five endometrial samples were obtained from fertile women and processed by a standard protocol to obtain human endometrial stromal cells for in vitro studies. Stromal cells were cultured in vitro and decidualized for 8 days. At day 6, copper was added to the treatment group or camptothecin as positive control for apoptosis until day 8. Five endometrial samples were used in each group. The aim of this study was to analyze the effect of copper in apoptosis and necrosis by flow cytometry, to visualize the apoptotic microtubule network during apoptosis by immunofluorescence, and finally to determine the gene expression profile of a panel of 192 genes related to endometrial receptivity and immune system by quantitative reverse transcription PCR (RT-qPCR). Copper, compared to the decidualized group, induced changes in the gene expression by an order of magnitude in 49 genes (42 up- and 9 downregulated). This alteration in the decidualization gene signature by copper includes 19 genes involved in the endometriosis pathology and others related to other gynecological disorders such as preeclampsia and infertility. Our results indicate that copper does not increase the apoptosis level induced by the decidualization treatment. However, copper alters the gene expression of some biomarkers of endometrial receptivity and immune response.

The leptin system during human endometrial receptivity and preimplantation development.

The leptin system is implicated in the regulation of body weight and reproductive function, acting at endocrine and paracrine levels. This ligand-receptor system is mandatory for embryonic implantation in rodents. Here, we investigate the expression pattern of total leptin receptor (OB-R(T)), the long form (OB-R(L)) and short isoforms HuB219.1 and HuB219.3 in the human endometrium. Furthermore, we studied leptin and OB-R(T) mRNA during human embryonic preimplantation development and the embryonic regulation of the endometrial OB-R(L). Leptin receptor expression and its isoforms increase in the luteal phase and peak in the late part. Leptin receptor was localized at the epithelial and glandular epithelium using in situ hybridization. Reverse transcription-nested PCR showed the presence of OB-R(T) mRNA at all the embryonic stages, whereas leptin mRNA was only detected at the blastocyst stage. The embryonic regulation of endometrial epithelial OB-R(L) and HuB219.3 was studied, and no impact was found. Finally, OB-R(L) was immunohistochemically localized in the human cytotrophoblast and maternal decidua. These findings suggest that secretory endometrium is a target tissue for leptin action, and oocytes and preimplantation embryos possess OB-R mRNA, indicating that leptin may be necessary for embryonic development. Furthermore, leptin mRNA is specifically expressed at the blastocysts stage, suggesting a function in the blastocyst-endometrial dialogue.

Endometrial gene expression in the window of implantation is altered in obese women especially in association with polycystic ovary syndrome.

OBJECTIVE: To determine whether luteal phase endometrial transcriptome is altered in obese women during the window of implantation (WOI), considering the presence of infertility, fat distribution and association with polycystic ovary syndrome (PCOS). DESIGN: Prospective study. SETTING: University-affiliated infertility clinic, between May 2007 and March 2009. PATIENT(S): One control group of women with normal weight (n=4), and four study groups of obese women (n=6 each one) according to the association with infertility, PCOS, and ovarian stimulation. INTERVENTION(S): The endometrium was biopsied 7 days after LH surge or hCG administration in 28 women. MAIN OUTCOME MEASURE(S): Endometrial gene expression during the WOI. RESULT(S): One hundred and fifty-one genes were dysregulated in obese groups compared with controls. This dysregulation was more pronounced when infertility was associated. The biologic processes of these genes belonged mainly to development and regulation of different biological functions such as transcription and biosynthesis. The molecular functions overrepresented were transcription and peptide receptor activity. The endometrium of obese women with PCOS showed dysregulated genes related to biologic processes such as development, morphogenesis, and the immune system, as well as different molecular functions such as protein binding, binding, growth factor activity, and carboxylic acid transmembrane transporter activity. Some of these genes have been previously related to implantation and unexplained infertility. CONCLUSION(S): Obese women present a different endometrial gene expression than controls during the WOI, which is more pronounced when infertility or polycystic ovary syndrome are associated.

Human endometrial side population cells exhibit genotypic, phenotypic and functional features of somatic stem cells.

During reproductive life, the human endometrium undergoes around 480 cycles of growth, breakdown and regeneration should pregnancy not be achieved. This outstanding regenerative capacity is the basis for women's cycling and its dysfunction may be involved in the etiology of pathological disorders. Therefore, the human endometrial tissue must rely on a remarkable endometrial somatic stem cells (SSC) population. Here we explore the hypothesis that human endometrial side population (SP) cells correspond to somatic stem cells. We isolated, identified and characterized the SP corresponding to the stromal and epithelial compartments using endometrial SP genes signature, immunophenotyping and characteristic telomerase pattern. We analyzed the clonogenic activity of SP cells under hypoxic conditions and the differentiation capacity in vitro to adipogenic and osteogenic lineages. Finally, we demonstrated the functional capability of endometrial SP to develop human endometrium after subcutaneous injection in NOD-SCID mice. Briefly, SP cells of human endometrium from epithelial and stromal compartments display genotypic, phenotypic and functional features of SSC.

Embryonic adhesion is not affected by endometrial leptin receptor gene silencing.

OBJECTIVE: In rodents, evidence suggests that the leptin system is mandatory for embryonic implantation. We aimed to investigate the functional relevance of the endometrial leptin receptor (OB-R) in the adhesion phase of human implantation. DESIGN: We used an in vitro model for embryonic adhesion, composed of a human endometrial cell line (HEC1-A) and B6C3F1 mouse embryos. The OB-R gene was silenced in a stable manner by RNA interference, and embryonic adhesion rates were analyzed. SETTING: Research laboratory at a university-affiliated center. INTERVENTION(S): RNA interference. MAIN OUTCOME MEASURE(S): Embryonic adhesion in cells treated with OB-R RNAi. RESULT(S): The OB-R shRNA-transfected cells exhibited up to 80% lower OB-R mRNA levels than those of cells nontransfected or transfected with scrambled shRNA. The OB-R protein was also highly diminished in the stable OB-R shRNA-transfected HEC1A cells, whereas OAS1 expression was similar in both nontransfected and transfected cells. Embryonic adhesion rate was similar in nontransfected (94%) and HEC1-A transfected cells with a scrambled sequence (94%) or with OB-R silencing sequence (92%). CONCLUSION(S): Knocking down the OB-R gene in a human endometrial cell line has no effect on the embryonic adhesion rate. Nevertheless, the functional relevance of this system can not be excluded in other phases of embryonic implantation, such as the invasion phase. Moreover, we describe a new approach to the functional analysis of candidate molecules implicated in blastocyst adhesion.

Effect of controlled ovarian hyperstimulation in IVF on endometrial gene expression profiles.

Controlled ovarian hyperstimulation (COH) used in IVF produces lower implantation rates per embryo transferred compared to natural cycles utilized in ovum donation, suggesting a suboptimal endometrial development. Endometrial receptivity has recently been investigated in natural menstrual cycles with the aid of microarray technology. The aim of this study is to investigate the impact of COH using urinary gonadotrophins with a long protocol with GnRH agonists without progesterone supplementation (similar to the natural cycle) on endometrial gene expression profiles during the window of implantation by comparing the profiles at day hCG + 7 of COH versus LH + 7 of a previous natural cycle in the same women. For this purpose we have used microarray technology by Affymetrix (GeneChip HG_U133A), which allows more than 22,000 genes to be tested simultaneously. Results were validated by semi-quantitative PCR and quantitative PCR experiments. We found that more than 200 genes showed a differential expression of more than 3-fold when COH and normal cycles were compared at hCG + 7 versus LH + 7. We simultaneously re-analysed the LH + 2 versus LH + 7 endometrial gene expression profiles in previous natural cycles in the same subject using this specific GeneChip, the results obtained were consistent with our own published results. This is the first time that gene expression profiles of the endometrium during COH are reported. The large degree of gene expression disturbance is surprising and highlights the need for further efforts to optimize COH protocols.

Gene expression profiling of human endometrial receptivity on days LH+2 versus LH+7 by microarray technology.

In humans, embryonic implantation and reproduction depends on the interaction of the embryo with the receptive endometrium. To gain a global molecular understanding of human endometrial receptivity, we compared gene expression profiles of pre-receptive (day LH+2) versus receptive (LH+7) endometria obtained from the same fertile woman (n = 5) in the same menstrual cycle in five independent experiments. Biopsies were analysed using the Affymetrix HG-U95A array, a DNA chip containing approximately 12,000 genes. Using the pre-defined criteria of a fold change >/=3 in at least four out of five women, we identified 211 regulated genes. Of these, 153 were up-regulated at LH+7 versus LH+2, whereas 58 were down-regulated. Amongst these 211 regulated genes, we identified genes that were known to play a role in the development of a receptive endometrium, and genes for which a role in endometrial receptivity, or even endometrial expression, has not been previously described. Validation of array data was accomplished by mRNA quantification by real time quantitative fluorescent PCR (Q-PCR) of three up-regulated [glutathione peroxidase 3 (GPx-3), claudin 4 (claudin-4) and solute carrier family 1 member 1 (SLC1A1)] genes in independent LH+2 versus LH+7 endometrial samples from fertile women (n = 3) and the three up-regulated genes throughout the menstrual cycle (n = 15). Human claudin-4 peaks specifically during the implantation window, whereas GPx-3 and SLC1A1 showed highest expression in the late secretory phase. In-situ hybridization (ISH) experiments showed that GPx-3 and SLC1A1 expression was restricted to glandular and luminal epithelial cells during the mid- and late luteal phase. The present work adds new and important data in this field, and highlights the complexity of studying endometrial receptivity even using global gene-expression analysis.