Genetic insights into biological mechanisms governing human ovarian ageing.

Reproductive longevity is essential for fertility and influences healthy ageing in women1,2, but insights into its underlying biological mechanisms and treatments to preserve it are limited. Here we identify 290 genetic determinants of ovarian ageing, assessed using normal variation in age at natural menopause (ANM) in about 200,000 women of European ancestry. These common alleles were associated with clinical extremes of ANM; women in the top 1% of genetic susceptibility have an equivalent risk of premature ovarian insufficiency to those carrying monogenic FMR1 premutations3. The identified loci implicate a broad range of DNA damage response (DDR) processes and include loss-of-function variants in key DDR-associated genes. Integration with experimental models demonstrates that these DDR processes act across the life-course to shape the ovarian reserve and its rate of depletion. Furthermore, we demonstrate that experimental manipulation of DDR pathways highlighted by human genetics increases fertility and extends reproductive life in mice. Causal inference analyses using the identified genetic variants indicate that extending reproductive life in women improves bone health and reduces risk of type 2 diabetes, but increases the risk of hormone-sensitive cancers. These findings provide insight into the mechanisms that govern ovarian ageing, when they act, and how they might be targeted by therapeutic approaches to extend fertility and prevent disease.

KDM4A regulates the maternal-to-zygotic transition by protecting broad H3K4me3 domains from H3K9me3 invasion in oocytes.

The importance of germline-inherited post-translational histone modifications on priming early mammalian development is just emerging1-4. Histone H3 lysine 9 (H3K9) trimethylation is associated with heterochromatin and gene repression during cell-fate change5, whereas histone H3 lysine 4 (H3K4) trimethylation marks active gene promoters6. Mature oocytes are transcriptionally quiescent and possess remarkably broad domains of H3K4me3 (bdH3K4me3)1,2. It is unknown which factors contribute to the maintenance of the bdH3K4me3 landscape. Lysine-specific demethylase 4A (KDM4A) demethylates H3K9me3 at promoters marked by H3K4me3 in actively transcribing somatic cells7. Here, we report that KDM4A-mediated H3K9me3 demethylation at bdH3K4me3 in oocytes is crucial for normal pre-implantation development and zygotic genome activation after fertilization. The loss of KDM4A in oocytes causes aberrant H3K9me3 spreading over bdH3K4me3, resulting in insufficient transcriptional activation of genes, endogenous retroviral elements and chimeric transcripts initiated from long terminal repeats during zygotic genome activation. The catalytic activity of KDM4A is essential for normal epigenetic reprogramming and pre-implantation development. Hence, KDM4A plays a crucial role in preserving the maternal epigenome integrity required for proper zygotic genome activation and transfer of developmental control to the embryo.

Tumor miRNA expression profile is related to vestibular schwannoma growth rate.

OBJECTIVE: Our objective was to investigate if the tumor microRNA (miRNA) expression profile was related to tumor growth rate. Growth-related miRNAs might be potential targets for future therapeutic intervention. MATERIAL AND METHODS: Tumor tissue was sampled during surgery of patients with a sporadic vestibular schwannoma. Tumor growth rate was determined by tumor measurement on the two latest pre-operative MRI scans. Tumor miRNA expression was analyzed using the Affymetrix Gene Chip® protocol, and CEL files were generated using GeneChip® Command Console® Software and normalized using Partek Genomics Suite 6.5. The CEL files were analyzed using the statistical software program R. Principal component analysis, affected gene ontology analysis, and analysis of miRNA expression fold changes were used for analysis of potential relations between miRNA expression profile and tumor growth rate. RESULTS AND CONCLUSION: Tumor miRNA expression is related to the growth rate of sporadic vestibular schwannomas. Rapid tumor growth is associated with deregulation of several miRNAs, including upregulation of miR-29abc, miR-19, miR-340-5p, miR-21, and miR-221 and downregulation of miR-744 and let-7b. Gene ontologies affected by the deregulated miRNAs included neuron development and differentiation, gene silencing, and negative regulation of various biological processes, including cellular and intracellular signaling and metabolism.

Human granulosa cells function as innate immune cells executing an inflammatory reaction during ovulation: a microarray analysis.

Ovulation has been compared to a local inflammatory reaction. We performed an in silico study on a unique, PCR validated, transcriptome microarray study to evaluate if known inflammatory mechanisms operate during ovulation. The granulosa cells were obtained in paired samples at two different time points during ovulation (just before and 36 hours after ovulation induction) from nine women receiving fertility treatment. A total of 259 genes related to inflammation became significantly upregulated during ovulation (2-80 fold, p<0.05), while specific leukocyte markers were absent. The genes and pathway analysis indicated NF-KB-, MAPK- and JAK/STAT signalling (p<1.0E-10) as the major pathways involved in danger recognition and cytokine signalling to initiate inflammation. Upregulated genes further encoded enzymes in eicosanoid production, chemo-attractants, coagulation factors, cell proliferation factors involved in tissue repair, and anti-inflammatory factors to resolve the inflammation again. We conclude that granulosa cells, without involvement from the innate immune system, can orchestrate ovulation as a complete sterile inflammatory reaction.

Temporal expression pattern of genes during the period of sex differentiation in human embryonic gonads.

The precise timing and sequence of changes in expression of key genes and proteins during human sex-differentiation and onset of steroidogenesis was evaluated by whole-genome expression in 67 first trimester human embryonic and fetal ovaries and testis and confirmed by qPCR and immunohistochemistry (IHC). SRY/SOX9 expression initiated in testis around day 40 pc, followed by initiation of AMH and steroidogenic genes required for androgen production at day 53 pc. In ovaries, gene expression of RSPO1, LIN28, FOXL2, WNT2B, and ETV5, were significantly higher than in testis, whereas GLI1 was significantly higher in testis than ovaries. Gene expression was confirmed by IHC for GAGE, SOX9, AMH, CYP17A1, LIN28, WNT2B, ETV5 and GLI1. Gene expression was not associated with the maternal smoking habits. Collectively, a precise temporal determination of changes in expression of key genes involved in human sex-differentiation is defined, with identification of new genes of potential importance.

Gene expression profiling in patients with polymyalgia rheumatica before and after symptom-abolishing glucocorticoid treatment.

BACKGROUND: The pathophysiology, including the impact of gene expression, of polymyalgia rheumatica (PMR) remains elusive. We profiled the gene expression in muscle tissue in PMR patients before and after glucocorticoid treatment. METHODS: Gene expression was measured using Affymetrix Human Genome U133 Plus 2.0 arrays in muscle biopsies from 8 glucocorticoid-naive patients with PMR and 10 controls before and after prednisolone-treatment for 14 days. For 14 genes, quantitative real-time PCR (qRT-PCR, n = 9 in both groups) was used to validate the microarray findings and to further investigate the expression of genes of particular interest. RESULTS: Prednisolone normalized erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) in PMR patients. A total of 165 putatively clinically relevant, differentially expressed genes were identified (cut-off: fold difference > ±1.2, difference of mean > 30, and p < 0.05); of these, 78 genes differed between patients and controls before treatment, 131 genes responded to treatment in a given direction only in patients, and 44 fulfilled both these criteria. In 43 of the 44 genes, treatment counteracted the initial difference. Functional clustering identified themes of biological function, including regulation of protein biosynthesis, and regulation of transcription and of extracellular matrix processes. Overall, qRT-PCR confirmed the microarray findings: Microarray-detected group differences were confirmed for 9 genes in 17 of 18 comparisons (same magnitude and direction of change); lack of group differences in microarray testing was confirmed for 5 genes in 8 of 10 comparisons. Before treatment, using qRT-PCR, expression of interleukin 6 (IL-6) was found to be 4-fold higher in patients (p < 0.05). CONCLUSIONS: This study identifies genes in muscle, the expression of which may impact the pathophysiology of PMR. Moreover, the study adds further evidence of the importance of IL-6 in the disease. Follow-up studies are needed to establish the exact pathophysiological relevance of the identified genes. The study was retrospectively listed on the ISRCTN registry with study ID ISRCTN69503018 and date of registration the 26th of July 2017.

Efficient Generation of Glucose-Responsive Beta Cells from Isolated GP2+ Human Pancreatic Progenitors.

Stem cell-based therapy for type 1 diabetes would benefit from implementation of a cell purification step at the pancreatic endoderm stage. This would increase the safety of the final cell product, allow the establishment of an intermediate-stage stem cell bank, and provide a means for upscaling ß cell manufacturing. Comparative gene expression analysis revealed glycoprotein 2 (GP2) as a specific cell surface marker for isolating pancreatic endoderm cells (PECs) from differentiated hESCs and human fetal pancreas. Isolated GP2+ PECs efficiently differentiated into glucose responsive insulin-producing cells in vitro. We found that in vitro PEC proliferation declines due to enhanced expression of the cyclin-dependent kinase (CDK) inhibitors CDKN1A and CDKN2A. However, we identified a time window when reducing CDKN1A or CDKN2A expression increased proliferation and yield of GP2+ PECs. Altogether, our results contribute tools and concepts toward the isolation and use of PECs as a source for the safe production of hPSC-derived ß cells.

Overcoming the bottleneck of platelet lysate supply in large-scale clinical expansion of adipose-derived stem cells: A comparison of fresh versus three types of platelet lysates from outdated buffy coat-derived platelet concentrates.

BACKGROUND: Platelet lysates (PL) represent a promising replacement for xenogenic growth supplement for adipose-derived stem cell (ASC) expansions. However, fresh platelets from human blood donors are not clinically feasible for large-scale cell expansion based on their limited supply. Therefore, we tested PLs prepared via three methods from outdated buffy coat-derived platelet concentrates (PCs) to establish an efficient and feasible expansion of ASCs for clinical use. METHODS: PLs were prepared by the freeze-thaw method from freshly drawn platelets or from outdated buffy coat-derived PCs stored in the platelet additive solution, InterSol. Three types of PLs were prepared from outdated PCs with platelets suspended in either (1) InterSol (not manipulated), (2) InterSol + supplemented with plasma or (3) plasma alone (InterSol removed). Using these PLs, we compared ASC population doubling time, cell yield, differentiation potential and cell surface markers. Gene expression profiles were analyzed using microarray assays, and growth factor concentrations in the cell culture medium were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: Of the three PL compositions produced from outdated PCs, removal of Intersol and resuspension in plasma prior to the first freezing process was overall the best. This specific outdated PL induced ASC growth kinetics, surface markers, plastic adherence and differentiation potentials comparable with PL from fresh platelets. ASCs expanded in PL from fresh versus outdated PCs exhibited different expressions of 17 overlapping genes, of which 10 were involved in cellular proliferation, although not significantly reflected by cell growth. Only minor differences in growth factor turnover were observed. CONCLUSION: PLs from outdated platelets may be an efficient and reliable source of human growth supplement allowing for large-scale ASC expansion for clinical use.

Gene expression, signal transduction pathways and functional networks associated with growth of sporadic vestibular schwannomas.

The objective of this study was to determine global gene expression in relation to Vestibular schwannomas (VS) growth rate and to identify signal transduction pathways and functional molecular networks associated with growth. Repeated magnetic resonance imaging (MRI) prior to surgery determined tumor growth rate. Following tissue sampling during surgery, mRNA was extracted from 16 sporadic VS. Double stranded cDNA was synthesized from the mRNA and used as template for in vitro transcription reaction to synthesize biotin-labeled antisense cRNA, which was hybridized to Affymetrix HG-U133A arrays and analyzed by dChip software. Differential gene expression was defined as a 1.5-fold difference between fast and slow growing tumors (><0.5 ccm/year), employing a p-value <0.01. Deregulated transcripts were matched against established gene ontology. Ingenuity Pathway Analysis was used for identification of signal transduction pathways and functional molecular networks associated with tumor growth. In total 109 genes were deregulated in relation to tumor growth rate. Genes associated with apoptosis, growth and cell proliferation were deregulated. Gene ontology included regulation of the cell cycle, cell differentiation and proliferation, among other functions. Fourteen pathways were associated with tumor growth. Five functional molecular networks were generated. This first study on global gene expression in relation to vestibular schwannoma growth rate identified several genes, signal transduction pathways and functional networks associated with tumor progression. Specific genes involved in apoptosis, cell growth and proliferation were deregulated in fast growing tumors. Fourteen pathways were associated with tumor growth. Generated functional networks underlined the importance of the PI3K family, among others.

Changes in Gene Expression during G-CSF-Induced Emergency Granulopoiesis in Humans.

Emergency granulopoiesis refers to the increased production of neutrophils in bone marrow and their release into circulation induced by severe infection. Several studies point to a critical role for G-CSF as the main mediator of emergency granulopoiesis. However, the consequences of G-CSF stimulation on the transcriptome of neutrophils and their precursors have not yet been investigated in humans. In this work, we examine the changes in mRNA expression induced by administration of G-CSF in vivo, as a model of emergency granulopoiesis in humans. Blood samples were collected from healthy individuals after 5 d of G-CSF administration. Neutrophil precursors were sorted into discrete stages of maturation by flow cytometry, and RNA was subjected to microarray analysis. mRNA levels were compared with previously published expression levels in corresponding populations of neutrophil precursors isolated from bone marrow of untreated, healthy individuals. One thousand one hundred and ten mRNAs were differentially expressed >2-fold throughout terminal granulopoiesis. Major changes were seen in pathways involved in apoptosis, cytokine signaling, and TLR pathways. In addition, G-CSF treatment reduced the levels of four of five measured granule proteins in mature neutrophils, including the proantibacterial protein hCAP-18, which was completely deficient in neutrophils from G-CSF-treated donors. These results indicate that multiple biological processes are altered to satisfy the increased demand for neutrophils during G-CSF-induced emergency granulopoiesis in humans.

MicroRNA Expression Profile in Conjunctival Melanoma.

PURPOSE: Conjunctival melanoma (CM) is a rare disease associated with considerable mortality. As opposed to cutaneous melanoma, the epigenetic mechanisms involved in the development of CM and other mucosal melanomas (MMs) are unclear. The purpose of this study was to identify tumor-specific and prognostic microRNA (miRNA) in CM and to compare the miRNA profile with that of MM. METHODS: Using microarray analysis (Affymetrix) we determined the miRNA expression profile in 40 CMs compared with 7 normal conjunctival samples. Changes in miRNA expression were associated with T stage, local recurrence, metastasis, and mortality. Furthermore, the expression of six fresh frozen tissue samples of CM was compared with that of four laryngeal and sinonasal MM. RESULTS: Our analysis revealed 24 upregulated and 1 downregulated miRNA in CM; several of these miRNAs have key functions in the pathogenesis and progression of cutaneous melanoma. Additionally, we identified seven upregulated miRNAs specific for stage-T1 and stage-T2 CM, whose expression was associated with increased tumor thickness (P = 0.007), and two upregulated miRNAs (miR-3687 and miR-3916) associated with an increased risk of local recurrence. No stage T3-specific miRNAs were identified. CONCLUSIONS: We identified differentially expressed and potentially prognostic miRNAs in CM. Furthermore, the miRNA expression pattern of CM resembled that in MM. The identification of these differentially expressed miRNAs provides an entry point for future functional studies of miRNAs as prognostic or therapeutic targets in CM and highlights the resemblance between CM, MM, and cutaneous melanoma.

Potassium Channel Interacting Protein 2 (KChIP2) is not a transcriptional regulator of cardiac electrical remodeling.

The heart-failure relevant Potassium Channel Interacting Protein 2 (KChIP2) augments CaV1.2 and KV4.3. KChIP3 represses CaV1.2 transcription in cardiomyocytes via interaction with regulatory DNA elements. Hence, we tested nuclear presence of KChIP2 and if KChIP2 translocates into the nucleus in a Ca(2+) dependent manner. Cardiac biopsies from human heart-failure patients and healthy donor controls showed that nuclear KChIP2 abundance was significantly increased in heart failure; however, this was secondary to a large variation of total KChIP2 content. Administration of ouabain did not increase KChIP2 content in nuclear protein fractions in anesthetized mice. KChIP2 was expressed in cell lines, and Ca(2+) ionophores were applied in a concentration- and time-dependent manner. The cell lines had KChIP2-immunoreactive protein in the nucleus in the absence of treatments to modulate intracellular Ca(2+) concentration. Neither increasing nor decreasing intracellular Ca(2+) concentrations caused translocation of KChIP2. Microarray analysis did not identify relief of transcriptional repression in murine KChIP2(-/-) heart samples. We conclude that although there is a baseline presence of KChIP2 in the nucleus both in vivo and in vitro, KChIP2 does not directly regulate transcriptional activity. Moreover, the nuclear transport of KChIP2 is not dependent on Ca(2+). Thus, KChIP2 does not function as a conventional transcription factor in the heart.

Competence Classification of Cumulus and Granulosa Cell Transcriptome in Embryos Matched by Morphology and Female Age.

OBJECTIVE: By focussing on differences in the mural granulosa cell (MGC) and cumulus cell (CC) transcriptomes from follicles resulting in competent (live birth) and non-competent (no pregnancy) oocytes the study aims on defining a competence classifier expression profile in the two cellular compartments. DESIGN: A case-control study. SETTING: University based facilities for clinical services and research. PATIENTS: MGC and CC samples from 60 women undergoing IVF treatment following the long GnRH-agonist protocol were collected. Samples from 16 oocytes where live birth was achieved and 16 age- and embryo morphology matched incompetent oocytes were included in the study. METHODS: MGC and CC were isolated immediately after oocyte retrieval. From the 16 competent and non-competent follicles, mRNA was extracted and expression profile generated on the Human Gene 1.0 ST Affymetrix array. Live birth prediction analysis using machine learning algorithms (support vector machines) with performance estimation by leave-one-out cross validation and independent validation on an external data set. RESULTS: We defined a signature of 30 genes expressed in CC predictive of live birth. This live birth prediction model had an accuracy of 81%, a sensitivity of 0.83, a specificity of 0.80, a positive predictive value of 0.77, and a negative predictive value of 0.86. Receiver operating characteristic analysis found an area under the curve of 0.86, significantly greater than random chance. When applied on 3 external data sets with the end-point outcome measure of blastocyst formation, the signature resulted in 62%, 75% and 88% accuracy, respectively. The genes in the classifier are primarily connected to apoptosis and involvement in formation of extracellular matrix. We were not able to define a robust MGC classifier signature that could classify live birth with accuracy above random chance level. CONCLUSION: We have developed a cumulus cell classifier, which showed a promising performance on external data. This suggests that the gene signature at least partly include genes that relates to competence in the developing blastocyst.

Cancers of unknown primary origin (CUP) are characterized by chromosomal instability (CIN) compared to metastasis of know origin.

BACKGROUND: Cancers of unknown primary (CUPs) constitute ~5% of all cancers. The tumors have an aggressive biological and clinical behavior. The aim of the present study has been to uncover whether CUPs exhibit distinct molecular features compared to metastases of known origin. METHODS: Employing genome wide transcriptome analysis, Linear Discriminant Analysis (LDA) and Quadratic Discriminant Analysis (QDA), we defined the putative origins of a large series of CUP and how closely related a particular CUP was to corresponding metastases of known origin. LDA predictions were subsequently used to define a universal CUP core set of differentially expressed genes, that by means of gene set enrichment analysis was exploited to depict molecular pathways characterizing CUP. RESULTS: The analyses show that CUPs are distinct from metastases of known origin. CUPs exhibit inconsistent expression of conventional cancer biomarkers and QDA derived outlier scores show that CUPs are more distantly related to their primary tumor class than corresponding metastases of known origin. Gene set enrichment analysis showed that CUPs display increased expression of genes involved in DNA damage repair and mRNA signatures of chromosome instability (CIN), indicating that CUPs are chromosome unstable compared to metastases of known origin. CONCLUSIONS: CIN may account for the uncommon clinical presentation, chemoresistance and poor outcome in patients with CUP and warrant selective diagnostic strategies and treatment.

p38 MAPK activation upregulates proinflammatory pathways in skeletal muscle cells from insulin-resistant type 2 diabetic patients.

Skeletal muscle is the key site of peripheral insulin resistance in type 2 diabetes. Insulin-stimulated glucose uptake is decreased in differentiated diabetic cultured myotubes, which is in keeping with a retained genetic/epigenetic defect of insulin action. We investigated differences in gene expression during differentiation between diabetic and control muscle cell cultures. Microarray analysis was performed using skeletal muscle cell cultures established from type 2 diabetic patients with a family history of type 2 diabetes and clinical evidence of marked insulin resistance and nondiabetic control subjects with no family history of diabetes. Genes and pathways upregulated with differentiation in the diabetic cultures, compared with controls, were identified using Gene Spring and Gene Set Enrichment Analysis. Gene sets upregulated in diabetic myotubes were associated predominantly with inflammation. p38 MAPK was identified as a key regulator of the expression of these proinflammatory gene sets, and p38 MAPK activation was found to be increased in the diabetic vs. control myotubes. Although inhibition of p38 MAPK activity decreased cytokine gene expression from the cultured diabetic myotubes significantly, it did not improve insulin-stimulated glucose uptake. Increased cytokine expression driven by increased p38 MAPK activation is a key feature of cultured myotubes derived from insulin-resistant type 2 diabetic patients. p38 MAPK inhibition decreased cytokine expression but did not affect the retained defect of impaired insulin action in the diabetic muscle cells.

Inhibition of Notch signaling alters the phenotype of orthotopic tumors formed from glioblastoma multiforme neurosphere cells but does not hamper intracranial tumor growth regardless of endogene Notch pathway signature.

BACKGROUND: Brain cancer stem-like cells (bCSC) are cancer cells with neural stem cell (NSC)-like properties found in the devastating brain tumor glioblastoma multiforme (GBM). bCSC are proposed a central role in tumor initiation, progression, treatment resistance and relapse and as such present a promising target in GBM research. The Notch signaling pathway is often deregulated in GBM and we have previously characterized GBM-derived bCSC cultures based on their expression of the Notch-1 receptor and found that it could be used as predictive marker for the effect of Notch inhibition. The aim of the present project was therefore to further elucidate the significance of Notch pathway activity for the tumorigenic properties of GBM-derived bCSC. METHODS: Human-derived GBM xenograft cells previously established as NSC-like neurosphere cultures were used. Notch inhibition was accomplished by exposing the cells to the gamma-secretase inhibitor DAPT prior to gene expression analysis and intracranial injection into immunocompromised mice. RESULTS: By analyzing the expression of several Notch pathway components, we found that the cultures indeed displayed different Notch pathway signatures. However, when DAPT-treated neurosphere cells were injected into the brain of immunocompromised mice, no increase in survival was obtained regardless of Notch pathway signature and Notch inhibition. We did however observe a decrease in the expression of the stem cell marker Nestin, an increase in the proliferative marker Ki-67 and an increased number of abnormal vessels in tumors formed from DAPT-treated, high Notch-1 expressing cultures, when compared with the control. CONCLUSION: Based on the presented results we propose that Notch inhibition partly induces differentiation of bCSC, and selects for a cell type that more strongly induces angiogenesis if the treatment is not sustained. However, this more differentiated cell type might prove to be more sensitive to conventional therapies.

IGF-I, IGF-II, and Insulin Stimulate Different Gene Expression Responses through Binding to the IGF-I Receptor.

Insulin and the insulin-like growth factors (IGF)-I and -II are closely related peptides important for regulation of metabolism, growth, differentiation, and development. The IGFs exert their main effects through the IGF-I receptor. Although the insulin receptor is the main physiological receptor for insulin, this peptide hormone can also bind at higher concentrations to the IGF-I receptor and exert effects through it. We used microarray gene expression profiling to investigate the gene expression regulated by IGF-I, IGF-II, and insulin after stimulation of the IGF-I receptor. Fibroblasts from mice, knockout for IGF-II and the IGF-II/cation-independent mannose-6-phosphate receptor, and expressing functional IGF-I but no insulin receptors, were stimulated for 4 h with equipotent saturating concentrations of insulin, IGF-I, and IGF-II. Each ligand specifically regulated a group of transcripts that was not regulated by the other two ligands. Many of the functions and pathways these regulated genes were involved in, were consistent with the known biological effects of these ligands. The differences in gene expression might therefore account for some of the different biological effects of insulin, IGF-I, and IGF-II. This work adds to the evidence that not only the affinity of a ligand determines its biological response, but also its nature, even through the same receptor.

Comparison of gene expression profiles in granulosa and cumulus cells after ovulation induction with either human chorionic gonadotropin or a gonadotropin-releasing hormone agonist trigger.

OBJECTIVE: To explore differences in follicle transcriptomes in patients having oocyte maturation with either a bolus of hCG or GnRHa. DESIGN: Cumulus cells (CC) and mural granulosa cells (MGC) were isolated from preovulatory follicles in patients undergoing controlled ovarian stimulation, prospectively randomized to GnRHa or hCG triggering. SETTING: University-based facilities for clinical services and research. PATIENT(S): Twenty women with indication for IVF or intracytoplasmic sperm injection treatment were randomly allocated to hCG or GnRH agonist (GnRHa) trigger. INTERVENTION(S): MGC and CC were collected from individual follicles in connection with oocyte retrieval. MAIN OUTCOME MEASURE(S): RNA was extracted, labeled, amplified, and hybridized on HumanGene1.0ST GeneChip Affymetrix array. Expression data were robust multichip average normalized and compared using Partek and Ingenuity software. Array data were confirmed with reverse transcription-polymerase chain reaction analysis. RESULT(S): Comparing the transcriptomes between the groups, 391 and 252 genes were differentially expressed (fold change >1.5) in CC and MGC, respectively. The enriched bionetworks showed that CC genes highly represented "lipid metabolism and small molecule biochemistry" (network score, 41), while in MGC, the top network was "cardiovascular development and function and cellular movement" (network score, 50). For both CC and MGC, the regulator analysis suggested LH as the upstream regulator for the difference observed. In CC, the LH receptor was more highly expressed after GnRHa trigger, while in MGC, genes involved in angiogenesis such as angiopoietin 1 and semaphorin 3A were down- and up-regulated, respectively, in GnRHa- as compared with hCG-triggered patients. CONCLUSION(S): The comparisons between somatic cell transcriptomes from GnRHa- and hCG-triggered follicles showed significant functional differences in both CC (steroidogenesis) and MGC (angiogenesis) compartments.

The dormant and the fully competent oocyte: comparing the transcriptome of human oocytes from primordial follicles and in metaphase II.

Oocytes become enclosed in primordial follicles during fetal life and remain dormant there until activation followed by growth and meiotic resumption. Current knowledge about the molecular pathways involved in oogenesis is incomplete. This study identifies the specific transcriptome of the human oocyte in the quiescent state and at the pinnacle of maturity at ovulation. In silico bioinformatic comparisons were made between the transcriptome of human oocytes from dormant primordial follicles and that of human metaphase II (MII) oocytes and granulosa cells and unique gene expression profiles were identified as well as functional and pathway enrichments associated with the oocytes from the two developmental hallmarks. A total of 729 genes were highly enriched in oocytes from primodial follicles and 1456 genes were highly enriched in MII oocytes (>10-fold, P < 0.001) representing functional categories such as cell cycle regulation, DNA protection and epigenetics, with representative genes validated by qPCR analysis. Dominating canonical pathways in the oocytes from primordial follicles were androgen, estrogen receptor, glucocorticoid receptor and PI3K/AKT signaling (P < 0.001). In the MII, mitotic roles of polo-like kinases, estrogen receptor, JAK/Stat signaling (P < 0.001) and the ERK/MAPK (P < 0.01) signaling were enriched. Some of the highly differentially expressed genes were completely new in human reproduction (CDR1, TLC1A, UHRF2) while other genes [ABO, FOLR1 (folate receptor), CHRNA3 (nicotine receptor)] may relate to clinical observations as diverse as premature ovarian failure, folic acid deficiency and smoking affecting female fertility. The in silico analysis identified novel reproduction-associated genes and highlighted molecular mechanisms and pathways associated with the unique functions of the human oocyte in its two extremes during folliculogenesis. The data provides a fundamental basis for future functional studies in regulation of human oogenesis.