Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 12 de 12
Filter
1.
Fertil Steril ; 102(1): 307-317.e7, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24837612

ABSTRACT

OBJECTIVE: To characterize the transcriptome of luminal epithelia (LE) of fertile secretory endometria and compare the results with those from glandular epithelia (GE). DESIGN: Endometrial samples were collected at 2 and 7 days after initial blood LH surge in separate menstrual cycles. LE were obtained with the use of laser microdissection. mRNA was amplified with the use of linear polymerase chain reaction and hybridized to Agilent 4×44 microarrays. Gene analysis was used to identify differentially expressed mRNAs. Immunohistochemistry was used to assess nine proteins. SETTING: One IVF clinic. PATIENT(S): Seven Caucasian fertile cycling women. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Cycle dating with the use of blood endocrinologic markers, microarrays of laser-microdissected LE, immunohistochemical analysis. RESULT(S): One hundred sixty-one (of 401) differentially expressed mRNAs in LE were identified from the metabolism pathway. Increased selective protein expression in LE at 7 days after initial LH surge was observed. LE mRNA expression was the converse of that in GE. The two cell types each had a different significant biologic pathway identified. CONCLUSION(S): Our results introduce a new concept that LE differentially expressed mRNAs are in the converse direction to that of GE, indicating different biologic processes despite the GE being continuous with the luminal monolayer. This probable distinction of biologic roles has not been noted previously. Further investigations must take cognizance of this observation.


Subject(s)
Endometrium/metabolism , Epithelial Cells/metabolism , Fertility , Menstrual Cycle , Female , Fertility/genetics , Gene Expression Profiling/methods , Genetic Markers , Humans , Immunohistochemistry , Laser Capture Microdissection , Menstrual Cycle/ethnology , Menstrual Cycle/genetics , Oligonucleotide Array Sequence Analysis , RNA, Messenger/metabolism , Time Factors , White People
2.
Behav Brain Res ; 257: 62-70, 2013 Nov 15.
Article in English | MEDLINE | ID: mdl-24055493

ABSTRACT

Two recent microarray and qRT-PCR studies showed that inbred Roman high- (RHA-I, low anxiety and frustration vulnerability) and low-avoidance (RLA-I, high anxiety and frustration vulnerability) rats, psychogenetically selected on the basis of their divergence in two-way avoidance performance, differed in basal whole-brain and hippocampal expression of genes related to neurotransmission, emotion, stress, aversive learning, and drug seeking behavior. We have extended these studies by analyzing strain differences in hippocampal gene expression following a frustrative experience involving reward downshift, i.e. instrumental successive negative contrast (iSNC), a phenomenon in which the sudden reduction of an expected reward induces frustration/anxiety. Food-deprived male Roman rats were exposed to a reduction in the amount of solid food presented in the goal of a straight alley (from 12 pellets in "training" trials - i.e. preshift trials- to 2 pellets in "frustration testing" trials - i.e. postshift trials-). The iSNC effect, as measured by response latencies in the "postshift" trials, appeared only in RLA-I rats (i.e. higher response latencies in the 12-2 RLA-I group as compared to the 2-2 RLA-I control group in postshift trials). Two and a half hours after the "postshift" behavioral test, hippocampi were removed and stored (-80°C) until analysis. Microarray analysis of these hippocampi showed that four differentially-expressed, and qRT-PCR-validated genes (TAAR2, THAP1, PKD2L1, NANOS), have relevance for brain function and behavior, including schizophrenia, depression, anxiety, and drug addiction, thus showing the usefulness of Roman strains as a genetic model for research on the neurogenetic basis of frustration.


Subject(s)
Anxiety/pathology , Frustration , Gene Expression Regulation/genetics , Gene Expression Regulation/physiology , Hippocampus/metabolism , Motivation/genetics , Animals , Anxiety/genetics , Avoidance Learning/physiology , Behavior, Animal , Conditioning, Operant/physiology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Food Deprivation , Male , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Rats , Rats, Inbred Strains , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Species Specificity , TRPP Cation Channels/genetics , TRPP Cation Channels/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism
3.
Reprod Sci ; 20(3): 308-17, 2013 Mar.
Article in English | MEDLINE | ID: mdl-22902743

ABSTRACT

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/ß-catenin, ERK/MAPK, transforming growth factor ß (TGF-ß), 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.


Subject(s)
Endometrium/physiology , Gene Expression Regulation , MicroRNAs/biosynthesis , Adult , Female , Gene Targeting/methods , Humans
4.
Fertil Steril ; 99(2): 508-17, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23102856

ABSTRACT

OBJECTIVE: To compare the accuracy and reproducibility of the endometrial receptivity array (ERA) versus standard histologic methods. DESIGN: A comparative prospective study (May 2008-May 2012). SETTING: University-affiliated infertility clinic. PATIENT(S): Eighty-six healthy oocyte donors, regularly cycling, aged 20-34 years with a body mass index (BMI) of 19-25 kg/m(2). INTERVENTION(S): Endometrial biopsies were collected throughout the menstrual cycle. For the accuracy study, 79 samples were grouped into two cohorts: the training set (n = 79) for ERA machine-learning training and dating, and a dating subset (n = 49) for comparison between histologic and ERA dating. For the reproducibility study, seven women underwent ERA testing and it was repeated in the same patients on the same day of their cycle 29-40 months later. MAIN OUTCOME MEASURE(S): Concordance of histologic and ERA dating related to LH as a reference, and interobserver variability between pathologists were statistically analyzed by the quadratic weighted Kappa index. The ERA reproducibility was tested and its gene expression visualized by principal component analysis. RESULT(S): For each pathologist, concordance against LH peak yielded values of 0.618 (0.446-0.791) and 0.685 (0.545-0.824). Interobserver variability between pathologists yielded a Kappa index of 0.622 (0.435-0.839). Concordance for ERA dating against LH peak showed a value of 0.922 (0.815-1.000). Reproducibility of the ERA test was 100% consistent. CONCLUSION(S): The ERA is more accurate than histologic dating and is a completely reproducible method for the diagnosis of endometrial dating and receptivity status.


Subject(s)
Artificial Intelligence , Biopsy/methods , Diagnosis, Computer-Assisted/methods , Endometrium/cytology , Endometrium/metabolism , Ovulation Detection/methods , Adult , Biomarkers/analysis , Female , Humans , Reproducibility of Results , Sensitivity and Specificity , Young Adult
5.
Fertil Steril ; 97(6): 1365-73.e1-2, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22480820

ABSTRACT

OBJECTIVE: To map the changes in messenger RNA (mRNA) and protein abundance during the window of implantation in specifically endometrial stromal and glandular epithelial cells obtained using laser microdissection microscopy (LDM). DESIGN: Endometrial samples were collected from two menstrual cycles at 2 and 7 days after first significant rise in blood LH, and separate cell populations were obtained using LDM. A new generation linear polymerase chain reaction (PCR) amplified the mRNA, which were hybridized to both Affymetrix U133 Plus2 and Agilent 4x44K microarrays followed by gene set analysis. Immunohistochemistry assessed protein expression between the two collection times. SETTING: In vitro fertilization clinic. PATIENT(S): Nine Caucasian, fertile, cycling women. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Cycle dating using blood markers; microarrays on laser microdissected glands and stroma; dual platform microarray confirmation; immunohistochemical analysis of cell cycle proteins. RESULT(S): The two microarray platforms showed concordance. During the window of implantation, a statistically significant network of 22 mRNA associated with the cell cycle was down-regulated. Immunohistochemistry identified altered localization in stroma. CONCLUSION(S): Microarrays demonstrated glands and stroma have distinct mRNA signatures, each dependent on the day of the cycle. We characterized two compartments of the receptive endometrium with a transcriptomic signature identifying regulation of only the cell cycle. Immunohistochemical analysis of cell cycle proteins identified a signature staining pattern of nuclear relocalization of a group of cyclins of stromal cells, which may be clinically applicable.


Subject(s)
Embryo Implantation/genetics , Endometrium/physiology , Epithelial Cells/physiology , Fertilization in Vitro , Oligonucleotide Array Sequence Analysis , Stromal Cells/physiology , Adult , Endometrium/cytology , Female , Fertility/genetics , Gene Expression/physiology , Humans , Luteal Phase/genetics , Luteinizing Hormone/blood , Young Adult
6.
Neurosci Lett ; 504(3): 265-70, 2011 Oct 31.
Article in English | MEDLINE | ID: mdl-21967962

ABSTRACT

Microarray technology was used to explore differences in brain gene expression under basal conditions in two strains of psychogenetically selected rats which differ in anxiety/stress responses, the inbred Roman High-(RHA-I) and Roman Low-(RLA-I) Avoidance rats. Microarray analysis detected 14 up-regulated and 24 down-regulated genes in RLA-I vs. RHA-I rats functionally related to neurobiological processes. The differentially expressed genes CAMKK2, CRHBP, EPHX2, HOMER3, NDN, PRL and RPL6 were selected for microarray validation using qRT-PCR. EPHX2, CAMKK2 (both up-regulated in RLA-I vs. RHA-I rats) and HOMER3 (down-regulated in RLA-I vs. RHA-I rats) showed a similar tendency and fold-change both in microarray and RT-PCR analyses; PRL (up-regulated in RLA-I vs. RHA-I rats), CRHBP and RPL6 (both down-regulated in RLA-I vs. RHA-I animals) showed a similar tendency but a different order of magnitude of change among experiments; finally, NDN was validated neither in tendency nor in magnitude of change.


Subject(s)
Avoidance Learning , Brain/metabolism , Gene Expression Regulation , Nerve Tissue Proteins/genetics , Rats, Inbred Strains/genetics , Animals , Anxiety/genetics , Calcium-Calmodulin-Dependent Protein Kinase Kinase/biosynthesis , Calcium-Calmodulin-Dependent Protein Kinase Kinase/genetics , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Epoxide Hydrolases/biosynthesis , Epoxide Hydrolases/genetics , Female , Gene Expression Profiling , Homer Scaffolding Proteins , Intracellular Signaling Peptides and Proteins/genetics , Nerve Tissue Proteins/biosynthesis , Oligonucleotide Array Sequence Analysis , Prolactin/biosynthesis , Prolactin/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Reverse Transcriptase Polymerase Chain Reaction , Ribosomal Proteins/biosynthesis , Ribosomal Proteins/genetics , Stress, Psychological/genetics
7.
Fertil Steril ; 96(4): 943-50, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21821247

ABSTRACT

OBJECTIVE: To evaluate the effect of adenomyosis on endometrial gene expression and its correlation with clinical outcome. DESIGN: Transcriptomic analysis of the endometrium of women with adenomyosis during the window of implantation. Retrospective matched cohort study of the impact of adenomyosis on oocyte donation (OD) outcome. SETTING: University-affiliated infertility clinic (2005-2009). PATIENT(S): Endometrial samples were analyzed using microarrays in women with adenomyosis and healthy controls. The clinical study included three groups: adenomyosis, endometriosis, and control. INTERVENTION(S): Endometrial biopsies in natural cycles 7 days after the LH peak; controlled ovarian stimulation in donors; ET in recipients after replacement therapy. MAIN OUTCOME MEASURE(S): Differentially expressed genes; implantation, pregnancy, miscarriage, and term pregnancy rates in OD. RESULT(S): There is a similar endometrial gene expression pattern in both the adenomyosis group and controls, and nonparametric tests revealed 34 dysregulated genes in adenomyosis patients but none belonged to the group of window of implantation genes. Implantation in OD did not differ among the three groups. However, miscarriage was significantly higher in the adenomyosis group vs. the adenomyosis + endometriosis and control groups. Term pregnancy rate was also significantly lower in the adenomyosis group compared with others. CONCLUSION(S): Clinical and molecular data show that implantation is not affected by adenomyosis, but the higher miscarriage rates associated with this condition lead to lower term pregnancy rates, indicating a clear negative effect on the final outcome of OD.


Subject(s)
Abortion, Spontaneous/etiology , Abortion, Spontaneous/pathology , Embryo Implantation/physiology , Endometriosis/complications , Endometriosis/diagnostic imaging , Oocyte Donation/methods , Abortion, Spontaneous/genetics , Adult , Cohort Studies , Endometriosis/genetics , Female , Gene Expression Profiling/methods , Humans , Pregnancy , Pregnancy Rate/trends , Retrospective Studies , Ultrasonography
8.
Fertil Steril ; 95(1): 50-60, 60.e1-15, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20619403

ABSTRACT

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.


Subject(s)
Endometrium/physiology , Gene Expression Profiling/methods , Genomics/methods , Uterine Diseases/diagnosis , Uterine Diseases/genetics , Cluster Analysis , Female , Gene Expression Profiling/standards , Genomics/standards , Humans , Infertility, Female/diagnosis , Infertility, Female/genetics , Menstrual Cycle/physiology , Predictive Value of Tests , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction/standards , Sensitivity and Specificity
9.
Zygote ; 17(1): 37-43, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19032801

ABSTRACT

Cycle synchronization of donor cells in the G0/G1 stage is a crucial step for successful somatic cell nuclear transfer. In the present report, we evaluated the effects of contact inhibition, serum starvation and the reagents - dimethyl sulphoxide (DMSO), roscovitine and cycloheximide (CHX) - on synchronization of canine fibroblasts at the G0/G1 stage. Ear fibroblast cells were collected from a beagle dog, placed into culture and used for analysis at passages three to eight. The population doubling time was 36.5 h. The proportion of G0/G1 cells was significantly increased by contact inhibition (77.1%) as compared with cycling cells (70.1%); however, extending the duration of culture did not induce further synchronization. After 24 h of serum starvation, cells were effectively synchronized at G0/G1 (77.1%). Although synchronization was further increased gradually after 24 h and even showed significant difference after 72 h (82.8%) of starvation, the proportion of dead cells also significantly increased after 24 h. The percentage of cells at the G0/G1 phase was increased (as compared with controls) after 72 h treatment with DMSO (76.1%) and after 48 h treatment with CHX (73.0%) or roscovitine (72.5%). However, the rate of cell death was increased after 24 and 72 h of treatment with DMSO and CHX, respectively. Thus, we recommend the use of roscovitine for cell cycle synchronization of canine ear fibroblasts as a preparatory step for SCNT.


Subject(s)
Ear/physiology , Fibroblasts/physiology , G1 Phase/physiology , Nuclear Transfer Techniques , Resting Phase, Cell Cycle/physiology , Animals , Antifungal Agents/pharmacology , Antineoplastic Agents/pharmacology , Contact Inhibition , Cycloheximide/pharmacology , Dimethyl Sulfoxide/pharmacology , Dogs , Female , Fibroblasts/drug effects , Free Radical Scavengers/pharmacology , G1 Phase/drug effects , Purines/pharmacology , Resting Phase, Cell Cycle/drug effects , Roscovitine , Serum
10.
J Clin Endocrinol Metab ; 93(9): 3490-8, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18559911

ABSTRACT

CONTEXT: Uterine leiomyomas are the most frequent benign tumors during reproductive age. Whether intramural leiomyomas cause infertility and should be removed is controversial because no study has addressed the underlying mechanism of infertility. OBJECTIVE: The objective of the study was to test the effect of intramural leiomyomas on endometrial function by comparing gene during the window of implantation and implantation in an oocyte donation program, in which the quality of the embryos replaced is similar and the endocrine environment of the endometrium is standardized by exogenous steroids. DESIGN: Human endometria of women with single intramural leiomyomas (group A, <5 cm and group B, > or =5 cm) and controls (group C) were collected on day LH+7 and processed for histology and gene expression analysis, using different methods and validated by quantitative RT-PCR. To compare in vitro fertilization outcome, a total of 1035 cases from our oocyte donation database were included, comprising patients with one fibroid less than 5 cm (A1, n = 532); two leiomyomas less than 5 cm (A2, n = 128); three or more leiomyomas less than 5 cm (A3, n = 125); one fibroid 5 cm or greater (B, n = 22); and two control groups: C1 (n = 93), women with previous myomectomy; and C2 (n = 135), women without uterine pathology treated on the same dates as C1. RESULTS: There was a strong positive and negative correlation in the expression profile of 69 genes according to the leiomyomas's size, but only three of the 25 genes related to the window of implantation were dysregulated. Term pregnancy rates after oocyte donation were 36.9, 34.1, 39.0, 36.4, 39.2, and 42.6% (P = 0.769) among the established groups. Similarly, no correlation between implantation and miscarriage with leiomyoma number and size was found. CONCLUSIONS: This study provides evidence that intramural leiomyomas not affecting the endometrial cavity alters the expression pattern of some endometrial genes, but the genes involved in implantation are not affected. This is confirmed by leiomyomas having no effect on oocyte donation outcome when the size and number of leiomyomas are analyzed.


Subject(s)
Embryo Implantation/genetics , Embryo Implantation/physiology , Endometrium/physiology , Gene Expression Regulation, Neoplastic , Leiomyoma/physiopathology , Pregnancy Complications, Neoplastic/physiopathology , Uterine Neoplasms/physiopathology , Adult , Cluster Analysis , Endometrium/metabolism , Female , Gene Expression Profiling , Genomics , Humans , Leiomyoma/genetics , Leiomyoma/metabolism , Middle Aged , Models, Biological , Oligonucleotide Array Sequence Analysis , Pregnancy , Pregnancy Complications, Neoplastic/genetics , Pregnancy Rate , Retrospective Studies , Uterine Neoplasms/genetics , Uterine Neoplasms/metabolism
11.
Fertil Steril ; 90(2): 450-2, 2008 Aug.
Article in English | MEDLINE | ID: mdl-17936281

ABSTRACT

MUC1 is a cell surface glycoprotein with a previously described antiadhesive role involved in different aspects of reproductive function. We found MUC1 expressed in male germ cell line and within the ejaculated sperm, but its presence in mature sperm does not seem to be related to male fertility. This was confirmed after analysis of results from assisted reproduction techniques with oocyte donation related with MUC1, although higher MUC1 expression is related to sperm recovery after preparation.


Subject(s)
Fertility/physiology , Mucin-1/analysis , Spermatozoa/chemistry , Testis/chemistry , Azoospermia/physiopathology , Ejaculation , Humans , Male , Mucin-1/physiology
12.
Reprod Biomed Online ; 15(1): 45-50, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17623534

ABSTRACT

Ovarian stimulation in assisted reproduction technology produces lower implantation rates per embryo transferred than natural and ovum donation cycles, suggesting suboptimal endometrial development due to the abnormal concentrations of hormones used to recruit more oocytes. After the publication of several studies on the gene expression profile of endometrial receptivity in the natural cycle using microarray technology, researchers have investigated the impact of ovarian stimulation on the gene expression pattern of the endometrium. Ovarian stimulation cycles that use gonadotrophin-releasing hormone (GnRH) agonists and antagonists have been analysed in detail during the window of implantation to establish differences compared with the natural cycle. This paper reviews results obtained in different studies to elucidate the changes induced by the different protocols used in clinics. At the morphological level, no relevant alteration was observed in endometrial development in the early and mid-luteal phases in women undergoing ovarian stimulation following GnRH antagonist treatments. However, the gene expression pattern of the endometrium showed some differences. In addition, the endometrial development after GnRH antagonist mimics the natural endometrium more closely than after GnRH agonist at both the morphological (no relevant differences) and molecular level (only 23 genes dysregulated at high dose). Clinical implications of these differences should be analysed in more detail.


Subject(s)
Endometrium/physiology , Genomics , Ovulation Induction/adverse effects , Uterine Diseases/etiology , Uterine Diseases/genetics , Female , Gene Expression Regulation , Humans
SELECTION OF CITATIONS
SEARCH DETAIL
...