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Swain J.E.,CCRM Inc | Carrell D.,University of Utah | Cobo A.,Instituto Valenciano Of Infertilidad | Meseguer M.,Instituto Valenciano Of Infertilidad | And 2 more authors.
Fertility and Sterility | Year: 2016

With increased use of comprehensive chromosome screening (CCS), the question remains as to why some practices do not experience the same high levels of clinical success after implementation of the approach. Indeed, the debate surrounding the efficacy and usefulness of blastocyst biopsy and CCS continues. Importantly, several variables impact the success of an assisted reproductive technology cycle. Transfer of a euploid embryo is but one factor in an intricate system that requires numerous steps to occur successfully. Certainly, the culture environment and the manipulations of the embryo during its time in the laboratory can impact its reproductive potential. Environmental stressors ranging from culture media to culture conditions and even culture platform can impact biochemical, metabolic, and epigenetic patterns that can affect the developing cell independent of chromosome number. Furthermore, accompanying procedures, such as biopsy and vitrification, are complex and, when performed improperly, can negatively impact embryo quality. These are areas that likely still carry room for improvement within the IVF laboratory. © 2016 American Society for Reproductive Medicine.

Bronet F.,IVI Madrid | Nogales M.-C.,IVI Madrid | Martinez E.,IVI Madrid | Ariza M.,IVI Madrid | And 3 more authors.
Fertility and Sterility | Year: 2015

Objective To study if it is possible to identify embryo sex from embryo cleavage timings. Design Retrospective and observational study. Setting University-affiliated private fertility center. Patient(s) Women undergoing preimplantion genetic diagnosis. Intervention[s) All biopsied embryos were cultured in an Embryoscope incubator with time-lapse technology. Main Outcome Measure(s) Cleavage timing from insemination to day 3 and all kinetic parameters that have been described in previous studies by our group. Result(s) The study included 421 embryos from our Compressive Chromosome Screening program, conducted from January 2012 to December 2012. Embryos were grouped according to their sex: male (176 embryos) and female (161 embryos). Chromosomal abnormal rate was similar for the two groups (male 62.5%, female 58.4%). When morphokinetic parameters were separated in different quartiles and grouped, we found statistical differences between male or female embryos. By logistic regression analysis we found that two specific kinetic variables were relevant: second synchrony (>2 hours) and timing of morula formation (80.8-90.9 hours). With the use of these parameters, we propose an algorithm with four different categories reflecting the range from 71% to 42% in the likelihood of an embryo being female. Conclusion(s) Embryo development was affected by embryo sex, and the sex ratio could be affected by the embryo selection method for transfer based on kinetic parameters. © 2015 American Society for Reproductive Medicine.

Krjutskov K.,Competence Center on Health Technologies | Krjutskov K.,Karolinska Institutet | Katayama S.,Karolinska Institutet | Saare M.,Competence Center on Health Technologies | And 13 more authors.
Human Reproduction | Year: 2016

STUDY QUESTION How can we study the full transcriptome of endometrial stromal and epithelial cells at the single-cell level? SUMMARY ANSWER By compiling and developing novel analytical tools for biopsy, tissue cryopreservation and disaggregation, single-cell sorting, library preparation, RNA sequencing (RNA-seq) and statistical data analysis. WHAT IS KNOWN ALREADY Although single-cell transcriptome analyses from various biopsied tissues have been published recently, corresponding protocols for human endometrium have not been described. STUDY DESIGN, SIZE, DURATION The frozen-thawed endometrial biopsies were fluorescence-activated cell sorted (FACS) to distinguish CD13-positive stromal and CD9-positive epithelial cells and single-cell transcriptome analysis performed from biopsied tissues without culturing the cells. We studied gene transcription, applying a modern and efficient RNA-seq protocol. In parallel, endometrial stromal cells were cultured and global expression profiles were compared with uncultured cells. PARTICIPANTS/MATERIALS, SETTING, METHODS For method validation, we used two endometrial biopsies, one from mid-secretory phase (Day 21, LH+8) and another from late-secretory phase (Day 25). The samples underwent single-cell FACS sorting, single-cell RNA-seq library preparation and Illumina sequencing. MAIN RESULTS AND THE ROLE OF CHANCE Here we present a complete pipeline for single-cell gene-expression studies, from clinical sampling to statistical data analysis. Tissue manipulation, starting from disaggregation and cell-type-specific labelling and ending with single-cell automated sorting, is managed within 90 min at low temperature to minimize changes in the gene expression profile. The single living stromal and epithelial cells were sorted using CD13- and CD9-specific antibodies, respectively. Of the 8622 detected genes, 2661 were more active in cultured stromal cells than in biopsy cells. In the comparison of biopsy versus cultured cells, 5603 commonly expressed genes were detected, with 241 significantly differentially expressed genes. Of these, 231 genes were up- and 10 down-regulated in cultured cells, respectively. In addition, we performed a gene ontology analysis of the differentially expressed genes and found that these genes are mainly related to cell cycle, translational processes and metabolism. LIMITATIONS, REASONS FOR CAUTION Although CD9-positive single epithelial cells sorting was successfully established in our laboratory, the amount of transcriptome data per individual epithelial cell was low, complicating further analysis. This step most likely failed due to the high dose of RNases that are released by the cells' natural processes, or due to rapid turnaround time or the apoptotic conditions in freezing- or single-cell solutions. Since only the cells from the late-secretory phase were subject to more focused analysis, further studies including larger sample size from the different time-points of the natural menstrual cycle are needed. The methodology also needs further optimization to examine different cell types at high quality. WIDER IMPLICATIONS OF THE FINDINGS The symbiosis between clinical biopsy and the sophisticated laboratory and bioinformatic protocols described here brings together clinical diagnostic needs and modern laboratory and bioinformatic solutions, enabling us to implement a precise analytical toolbox for studying the endometrial tissue even at the single-cell level. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

Diaz-Gimeno P.,University of Valencia | Horcajadas J.A.,IGENOMIX | Martinez-Conejero J.A.,IGENOMIX | Esteban F.J.,University of Jaen | And 4 more authors.
Fertility and Sterility | Year: 2011

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. © 2011 American Society for Reproductive Medicine, Published by Elsevier Inc.

Sabariego M.,University of Jaen | Moron I.,University of Granada | Gomez M.J.,University of Jaen | Donaire R.,University of Jaen | And 5 more authors.
Behavioural Brain Research | Year: 2013

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. © 2013 Elsevier B.V.

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