J.A. Horcajadas

A GENOMIC DIAGNOSTIC TOOL FOR HUMAN ENDOMETRIAL RECEPTIVITY BASED ON THE TRANSCRIPTOMIC SIGNATURE

OBJECTIVE To create a genomic tool composed of a customized microarray and a bioinformatic predictor for endometrial dating and to detect pathologies of endometrial origin. To define the transcriptomic signature of human endometrial receptivity. DESIGN Two cohorts of endometrial samples along the menstrual cycle were used: one to select the genes to be included in the customized microarray (endometrial receptivity array [ERA]), and the other to be analyzed by ERA to train the predictor for endometrial dating and to define the transcriptomic signature. A third cohort including pathological endometrial samples was used to train the predictor for pathological classification. SETTING Healthy oocyte donors and patients. PATIENT(S) Healthy fertile women (88) and women with implantation failure (5) or hydrosalpinx (2). INTERVENTION(S) Human endometrial biopsies. MAIN OUTCOME MEASURE(S) The gene expression of endometrial biopsies. RESULT(S) The ERA included 238 selected genes. The transcriptomic signature was defined by 134 genes. The predictor showed a specificity of 0.8857 and sensitivity of 0.99758 for endometrial dating, and a specificity of 0.1571 and a sensitivity of 0.995 for the pathological classification. CONCLUSION(S) This diagnostic tool can be used clinically in reproductive medicine and gynecology. The transcriptomic signature is a potential endometrial receptivity biomarkers cluster.

Endometrial receptivity is affected in women with high circulating progesterone levels at the end of the follicular phase: a functional genomics analysis

BACKGROUND Elevated serum progesterone levels at the end of the follicular phase in controlled ovarian stimulation (COS) leads to a poorer ongoing pregnancy rate in IVF cycles due to reduced endometrial receptivity. The objective of this study was to use microarray technology to compare endometrial gene expression profiles at the window of implantation according to the levels of circulating progesterone. METHODS For this prospective cohort study, microarray data were obtained from endometrial biopsies from 12 young healthy oocyte donors undergoing COS with pituitary suppression by either gonadotrophin-releasing hormone (GnRH) agonists or antagonists, and recombinant FSH. On the day of recombinant chorionic gonadotrophin (rCG) administration, six women had serum progesterone levels (P) >1.5 ng/ml (study group) and six had serum P levels <1.5 ng/ml (control group). Endometrial samples were collected using a Pipelle catheter 7 days after the rCG injection. RESULTS Using the parametric test, we identified 140 genes significantly dysregulated (64 up- and 76 down-regulated) in the study group endometria compared with the control endometria, regardless of the GnRH analogue employed. These genes are related to cell adhesion, developmental processes, the immune system and others, which are all required for normal endometrial function development. Of the 25 gene targets previously proposed as markers for endometrial receptivity, 13 appeared over-regulated in the study group. CONCLUSIONS Our results reveal that elevated progesterone levels on the day of rCG administration can induce significant alterations in the gene expression profile of the endometrium.

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 womens 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.

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.

Endometrial gene expression analysis at the time of embryo implantation in women with unexplained infertility

Successful embryo implantation depends on the quality of the embryo, as well as on the receptivity of the endometrium. The aim of this study was to investigate the endometrial gene expression profile in women with unexplained infertility in comparison with fertile controls at the time of embryo implantation in order to find potential predictive markers of uterine receptivity and to identify the molecular mechanisms of infertility. High-density oligonucleotide gene arrays, comprising 44 000 gene targets, were used to define the endometrial gene expression profile in infertile (n = 4) and fertile (n = 5) women during the mid-secretory phase (day LH + 7). Microarray results were validated using real-time PCR. Analyses of expression data were carried out using non-parametric methods. Hierarchical clustering and principal component analysis showed a clear distinction in endometrial gene expression between infertile and fertile women. In total we identified 145 significantly (>3-fold change) up-regulated and 115 down-regulated genes in infertile women versus controls. Via Database for Annotation, Visualization and Integrated Discovery functional analysis we detected a substantial number of dysregulated genes in the endometria of infertile women, involved in cellular localization (21.1%) and transport (18.8%) and transporter activity (13.1%) and with major localization in extracellular regions (19.2%). Ingenuity Pathways Analysis of the gene list showed dysregulation of gene pathways involved in leukocyte extravasation signalling, lipid metabolism and detoxification in the endometria of infertile women. In conclusion, endometrial gene expression in women with unexplained infertility at the time of embryo implantation is markedly different from that in fertile women. These results provide new information on genes and pathways that may have functional significance as regards to endometrial receptivity and subsequent embryo implantation.

Microarray analysis in sperm from fertile and infertile men without basic sperm analysis abnormalities reveals a significantly different transcriptome

Sperm analysis following World Health Organization guidelines is unable to explain the molecular causes of male infertility when basic sperm parameters are within a normal range and women do not present gynecologic pathology. Consequently, there is a need for accurate diagnostic tools in this area, and microarray technology emerges as promising. We present, for the first time, preliminary results of a comparison of sperm mRNA expression profiles between fertile and infertile men with normal semen parameters, discovering profound discrepancies between groups, with potential diagnostic and therapeutic possibilities.

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.

Predicting adverse obstetric outcome after early pregnancy events and complications: a review

BACKGROUND The aim was to evaluate the impact of early pregnancy events and complications as predictors of adverse obstetric outcome. METHODS We conducted a literature review on the impact of first trimester complications in previous and index pregnancies using Medline and Cochrane databases covering the period 1980-2008. RESULTS Clinically relevant associations of adverse outcome in the subsequent pregnancy with an odds ratio (OR) > 2.0 after complications in a previous pregnancy are the risk of perinatal death after a single previous miscarriage, the risk of very preterm delivery (VPTD) after two or more miscarriages, the risk of placenta praevia, premature preterm rupture of membranes, VPTD and low birthweight (LBW) after recurrent miscarriage and the risk of VPTD after two or more termination of pregnancy. Clinically relevant associations of adverse obstetric outcome in the ongoing pregnancy with an OR > 2.0 after complications in the index pregnancy are the risk of LBW and very low birthweight (VLBW) after a threatened miscarriage, the risk of pregnancy-induced hypertension, pre-eclampsia, placental abruption, preterm delivery (PTD), small for gestational age and low 5-min Apgar score after detection of an intrauterine haematoma, the risk of VPTD and intrauterine growth restriction after a crown-rump length discrepancy, the risk of VPTD, LBW and VLBW after a vanishing twin phenomenon and the risk of PTD, LBW and low 5-min Apgar score in a pregnancy complicated by severe hyperemesis gravidarum. CONCLUSIONS Data from our literature review indicate, by finding significant associations, that specific early pregnancy events and complications are predictors for subsequent adverse obstetric and perinatal outcome. Though, some of these associations are based on limited or small uncontrolled studies. Larger population-based controlled studies are needed to confirm these findings. Nevertheless, identification of these risks will improve obstetric care.

MicroRNAs miR-30b, miR-30d, and miR-494 Regulate Human Endometrial Receptivity

MicroRNAs (miRNAs) act as important epigenetic posttranscriptional regulators of gene expression. We aimed to gain more understanding of the complex gene expression regulation of endometrial receptivity by analyzing miRNA signatures of fertile human endometria. We set up to analyze miRNA signatures of receptive (LH + 7, n = 4) versus prereceptive (LH + 2, n = 5) endometrium from healthy fertile women. We found hsa-miR-30b and hsa-miR-30d to be significantly upregulated, and hsa-miR-494 and hsa-miR-923 to be downregulated in receptive endometrium. Three algorithms (miRanda, PicTar, and TargetScan) were used for target gene prediction. Functional analyses of the targets using Ingenuity Pathways Analysis and The Database for Annotation, Visualization and Integrated Discovery indicated roles in transcription, cell proliferation and apoptosis, and significant involvement in several relevant pathways, such as axon guidance, Wnt/β-catenin, ERK/MAPK, transforming growth factor β (TGF-β), p53 and leukocyte extravasation. Comparison of predicted miRNA target genes and our previous messenger RNA microarray data resulted in a list of 12 genes, including CAST, CFTR, FGFR2, and LIF that could serve as a panel of genes important for endometrial receptivity. In conclusion, we suggest that a subset of miRNAs and their target genes may play important roles in endometrial receptivity.