Granulosa cell responses to Gonal vs Menopur
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE16523 | GEO | 2010/03/22
SECONDARY ACCESSION(S): PRJNA116085
REPOSITORIES: GEO
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Project description:Goal of the experiment: To examine differential gene expression in granulosa cells of women undergoing ovarian stimulation with either recombinant human follicle stimulating hormone or purified human menopausal gonadotropin for in vitro fertilization. Brief description of the experiment: The study was designed to test the hypothesis that human granulosa cell (GC) gene expression response differs between pure recombinant FSH and human menopausal gonadotropin (hMG) stimulation regimens. Women < 35 years-old undergoing in vitro fertilization for tubal or male factor infertility were prospectively randomized to one of two stimulation protocols, Gonadotropin releasing hormone (GnRH) agonist long protocol plus individualized dosages of (1) recombinant follicle stimulating hormone (rFSH) (Gonal-F®) (n=4) or (2) purified human menopausal gonadotropin (hMG; Menopur®; 75 IU FSH/75 IU LH per vial) (n=3). Follicle development was monitored by ultrasound and serum estradiol levels. When two follicles were ≥ 17mm, human chorionic gonadotropin (hCG) (10,000 IU; Novarel®) was administered. Oocytes were retrieved 35 h post-hCG. Following oocyte recovery, GC were immediately collected in RNALater®. Pooled GC from individual patients were washed, centrifuged over 40% Percoll®, resuspended in RNALater, and snap-frozen in LN. Total RNA was extracted and stored at -70C. Biotinylated cRNA was synthesized from total RNA and each sample was run individually on CodeLink Whole Human Genome Bioarrays (Applied Microarrays). Unnamed genes and genes with <2-fold difference in expression were excluded from further analysis. After exclusions, 1736 genes exhibited differential expression between groups. Over 400 were categorized as signal transduction genes, ~180 as transcriptional regulators, and ~175 as enzymes/metabolic genes. Expression of selected genes was confirmed by RT-PCR. Differentially expressed genes included A kinase anchor protein 11 (AKAP11), bone morphogenic protein receptor II (BMPR2), epidermal growth factor (EGF), insulin-like growth factor binding protein (IGFBP)-4, IGFBP-5, basigin, and hypoxia-inducible factor (HIF)-1 alpha. The results suggest that major differences exist in the mechanism by which pure FSH alone versus FSH/LH regulate gene expression in preovulatory GC that could impact oocyte maturity and developmental competence. Experimental design: Women < 35 years-old undergoing in vitro fertilization for tubal or male factor infertility were prospectively randomized to one of two stimulation protocols, Gonadotropin releasing hormone (GnRH) agonist long protocol plus individualized dosages of (1) recombinant follicle stimulating hormone (rFSH) (Gonal-F®) (n=4) or (2) purified human menopausal gonadotropin (hMG; Menopur®; 75 IU FSH/75 IU LH per vial) (n=3). Follicle development was monitored by ultrasound and serum estradiol levels. When two follicles were ≥ 17mm, human chorionic gonadotropin (hCG) (10,000 IU; Novarel®) was administered. Oocytes were retrieved 35 h post-hCG. Following oocyte recovery, GC were immediately collected in RNALater®. Pooled GC from individual patients were washed, centrifuged over 40% Percoll®, resuspended in RNALater, and snap-frozen in LN. Total RNA was extracted and stored at -70C. Biotinylated cRNA was synthesized from total RNA and each sample was run individually on CodeLink Whole Human Genome Bioarrays (Applied Microarrays Quality control steps: The cRNA that was synthesized from the granulosa cells from individual patients was used for hybridization to a single CodeLink (Applied Microarrays) Whole Human Genome microarray. Only one sample was hybridized with each slide and only one dye (Alexa 647) was used so no dye swaps were necessary. Bacterial control spikes were used as per manufacturer's instructions. Samples used, extract preparation and labelling: The origin of each biological sample: Granulosa cells from women < 35 years-old undergoing in vitro fertilization. Manipulations of biological samples and protocols used: Female patients were treated with gonadotropin releasing hormone (GnRH) agonist long protocol plus individualized dosages of (1) recombinant follicle stimulating hormone (rFSH) (Gonal-F®) (n=4) or (2) purified human menopausal gonadotropin (hMG; Menopur®; 75 IU FSH/75 IU LH per vial) (n=3). Follicle development was monitored by ultrasound and serum estradiol levels. When two follicles were ≥ 17mm, human chorionic gonadotropin (hCG) (10,000 IU; Novarel®) was administered. Oocytes were retrieved 35 h post-hCG. Following oocyte recovery, granulosa cells were immediately collected in RNALater®. Pooled GC from individual patients were washed, centrifuged over 40% Percoll®, resuspended in RNALater, and snap-frozen in LN. Experimental factor: treatment with recombinant FSH versus purified human menopausal gonadotropin Technical protocols: Granulosa cells were collected into RNAlater (Ambion) and stored at -80C until processed. The granulosa cells were pelleted by centrifugation to remove the RNAlater. Each cell pellet was homogenized in TRI reagent (Molecular Research Centers). Bromochloropropane and sodium acetate were added, and the samples were centrifuged to separate the phases. The RNA-containing layer was removed and the RNA purified on an RNeasy extraction column (Qiagen). The sample was treated with an on-column DNase treatment (RNase-free DNase, Qiagen). The purity and quantity were evaluated by an Agilent Bioanalyzer using the RNA 6000 Nanoassay LabChip. Labelled cRNA was prepared using the manufacturer's Instruction Protocol (www1.amershambiosciences.com, CodeLink Gene Expression System: Manual Labelled cRNA Target Preparation). The source of reagents was the CodeLink Expression Assay Reagent Kit, Manual Prep, except where specified otherwise. 0.285 micrograms of total granulosa cell RNA was mixed with bacterial control RNA spikes and primed with T7 oligo(dT) primer for 10 min at 70C. (The bacterial control spikes included araB, entF, fixB, gnd, hisB, and leuB.) The first strand of cDNA was synthesized with first strand buffer, dNTP mix, RNase inhibitor, and reverse transcriptase for 2 h at 42C. The second strand cDNA synthesis reaction was prepared using second strand buffer, dNTP mix, DNA polymerase mix, and RNase H; the reaction was carried out for 2h at 16C. The double-stranded cDNA was purified on QIAquick columns (Qiagen) and the eluent was dried down in a SpeedVac concentrator. The double-stranded cDNA was resuspended in a mixture containing T7 reaction buffer, T7 ATP, T7 GTP, T7 UTP, T7 CTP, biotin-11-UTP, and T7 enzyme mix for the synthesis of cRNA. The cRNA synthesis reaction was terminated after 14h at 37C by purifying the cRNA on RNeasy columns (Qiagen). The concentration of cRNA was determined by spectrophotometry. Hybridization procedures and parameters: 10 micrograms of cRNA was mixed with fragmentation buffer and heated to 94C for 20 min. The fragmented cRNA was mixed with CodeLink hybridization buffer, loaded on the microarray slides, and hybridized for 18 hours at 37C. The slides were washed in 0.75x TNT (Tris-HCl, NaCl, Tween-20) at 46C for 1h then incubated with streptavidin-Alexa 647 fluorescent dye for 30 min at room temperature. The Alexa fluor was prepared in TNB blocking buffer (0.1M Tris-HCl, 0.15M NaCL, 0.5% NEN Blocking Reagent-PerkinElmer) The slides were then washed 4 times for 5 min each in 1x TNT and once in 0.05% Tween 20 for 5 sec. The slides were dried by centrifugation and scanned in an Axon GenePix 4000B scanner.
2010-03-22 | E-GEOD-16523 | biostudies-arrayexpress
Project description:Follicle stimulating hormone (FSH) and epidermal growth factor (EGF) are currently used on cumulus-oocyte complexes to mimic the luteinizing hormone surge in vitro and induce oocyte maturation and cumulus expansion. We have previously shown that addition of exogenous recombinant growth differentiation factor 9 (GDF9) during oocyte in vitro maturation led to an improvement of oocyte quality, as shown by an increased blastocyst percentage and fetal survival. Our objective was to characterize the effect of FSH/EGF and GDF9 treatments on mouse cumulus cells expression profile by microarray analysis. Cumulus-oocyte complexes (COCs) were recovered from 21 to 26 day old female 129/SV mice, 44 hours post equine chorionic gonadotropin treatment (eCG (5 IU)). For the microarray experiment whole COCs were treated with 293H control medium (0.125% v/v), with 20 ng/ml GDF9 or with a combination of 50 mIU/ml FSH and 10 ng/ml EGF. After 8 hours of in vitro maturation, COCs were denuded by gentle pipetting, the oocytes were removed and the cumulus cells centrifuged and extracted RNA analysed by microarray.
2011-12-10 | E-GEOD-19327 | biostudies-arrayexpress
Project description:The process of ovulation includes oocyte meiotic maturation, follicle rupture and transformation of the follicle into a corpus luteum. These events are initiated by the midcycle surge of gonadotropins and require the coordinated regulation of thousands of genes. The aim of the study was to monitor the changes in granulosa cell gene expression across five different time points during the first 36 hours of ovulation until follicle rupture, in order to increase our understanding of the events of human ovulation. We conducted a prospective cohort study including women undergoing ovarian stimulation for fertility treatment. Women were treated in a standard antagonist protocol with individually dosed human menopausal gonadotropin (hMG) or recombinant follicle stimulating hormone (rFSH). Ovulation was induced with either recombinant hCG (rhCG) or gonadotropin releasing hormone agonist (GNRHa). The granulosa cells were collected by transvaginal ultrasound-guided follicle puncture of one follicle at two specific time points during ovulation (repeated measurements), and the study covered a total of five time points: before ovulation induction (OI), 12, 17, 32 and 36 hours after OI.
2020-03-01 | GSE133868 | GEO
Project description:The objective of this study was to investigate the differences in the gene expression profile of the granulosa cells from preovulatory follicles obtained after controlled ovarian hyperstimulation (COH) with either recombinant FSH (rFSH) or urine derived human menopausal gonadotropins (hMG).
2008-01-31 | E-MEXP-1238 | biostudies-arrayexpress
Project description:To evaluate the mechanisms by which excessive FSH doses during ovarian stimulation are detrimental to ovarian follicle function and assisted reproduction outcomes, small ovarian reserve (SOR) heifers were subjected to ovarian stimulation with either a standard, 70 IU, or excessive, 210 IU, dose of commercial porcine FSH (cpFSH) preparation, per injection. The ovaries were recovered at the end of the ovarian stimulation protocol, consisting of eight cpFSH injections at 12 h intervals. In excessive dose treated heifers, heterogeneity was observed with regards to follicle phenotype including cumulus cell-oocyte complex (COC) morphology and intrafollicular steroid hormone milieu. In contrast, standard-dose treated heifers exhibited one predominant follicle phenotype which was selected as the control follicle type for all comparisons herein. RNA-seq analyses of granulosa, cumulus cells and the oocyte from individual follicles of these defined phenotypes was used to identify changes in the transcriptomes of these cell types in excessive dose treated individuals.
2022-08-10 | GSE197116 | GEO
Project description:Inhibin α knockout (Inha-/-) female mice develop sex cord-stromal ovarian cancer with complete penetrance and previous studies demonstrate that the pituitary gonadotropins [follicle stimulating hormone (FSH) and luteinizing hormone (LH)] are influential modifiers of granulosa cell tumor development and progression in inhibin-deficient females. Recent studies have demonstrated that Inha-/- ovarian follicles develop precociously to the early antral stage in prepubertal mice without any increase in serum FSH and these studies suggested that in the absence of inhibins, granulosa cells differentiate abnormally, and thus at sexual maturity may undergo an abnormal response to gonadotropin signaling. To test this hypothesis, we stimulated immature WT and Inha-/- female mice prior to gross tumor formation with gonadotropin analogs, and subsequently examined post-gonadotropin induced ovarian follicle development, as well as preovulatory and hCG-induced gene expression changes in granulosa cells. We find that at three weeks of age, inhibin-deficient ovaries do not show further antral development nor undergo cumulus expansion. Widespread alterations in the transcriptome of gonadotropin-treated Inha-/- granulosa cells suggest that gonadotropins initiate an improper program of cell differentiation in Inha-/- cells. Overall, our experiments reveal that inhibins are essential for the normal gonadotropin-dependent response of granulosa cells. (2) Genotypes (WT, Inh KO) collected from 2 preovulatory granulosa cells with and without hCG, in triplicate independent samples
2011-02-01 | E-GEOD-20466 | biostudies-arrayexpress
Project description:Ovulatory dysfunction affects nearly 40% of women of reproductive age that are struggling with infertility. Ovulation is the culmination of a complex and long process during which most follicles undergo atresia instead of developing into preovulatory follicles. The rescue of follicles from atresia and their maturation towards ovulatory competence requires “a pas de deux” between endocrine and locally-produced factors. Unequivocally, the most crucial endocrine factor is follicle-stimulating hormone (FSH). Insulin-like growth factors (IGFs) are among the most important local factors and are an irreplaceable partner of FSH. Therefore, understanding the mechanisms involved in the regulation of granulosa cell (GC) differentiation by FSH and IGF in humans will contribute to improve infertility treatments. In this report, we characterize a novel experimental approach to study human GC differentiation using cumulus cells from patients undergoing in vitro fertilization (IVF). Our findings demonstrate that human cumulus cells from IVF patients respond to FSH with the expression of genes that are known to be markers of granulosa cell differentiation during follicle maturation from the preantral to the large antral stage. Moreover, these results demonstrate that a large number of FSH-regulated genes require IGF1R activity and suggest that several aspects of follicle growth are coordinately regulated by FSH and the IGF system in humans. We used microarrays to detail the global effects of FSH on gene expression and to determine the effect that IGF1R inhibition has on FSH-induced stimulation.
2016-09-06 | GSE86427 | GEO
Project description:Inhibin α knockout (Inha-/-) female mice develop sex cord-stromal ovarian cancer with complete penetrance and previous studies demonstrate that the pituitary gonadotropins [follicle stimulating hormone (FSH) and luteinizing hormone (LH)] are influential modifiers of granulosa cell tumor development and progression in inhibin-deficient females. Recent studies have demonstrated that Inha-/- ovarian follicles develop precociously to the early antral stage in prepubertal mice without any increase in serum FSH and these studies suggested that in the absence of inhibins, granulosa cells differentiate abnormally, and thus at sexual maturity may undergo an abnormal response to gonadotropin signaling. To test this hypothesis, we stimulated immature WT and Inha-/- female mice prior to gross tumor formation with gonadotropin analogs, and subsequently examined post-gonadotropin induced ovarian follicle development, as well as preovulatory and hCG-induced gene expression changes in granulosa cells. We find that at three weeks of age, inhibin-deficient ovaries do not show further antral development nor undergo cumulus expansion. Widespread alterations in the transcriptome of gonadotropin-treated Inha-/- granulosa cells suggest that gonadotropins initiate an improper program of cell differentiation in Inha-/- cells. Overall, our experiments reveal that inhibins are essential for the normal gonadotropin-dependent response of granulosa cells.
2011-02-01 | GSE20466 | GEO
Project description:Background: With the rapid development of assisted reproductive technology (ART), although ovulation induction has made great progress, the accompanying health risks should not be ignored. Epigenetic modifications play an important role during the development of gametes and embryos. Recently, the epigenetic abnormalities caused by superovulation have attracted increasing attention. The follicle-stimulating hormone (FSH) and human menopausal gonadotropin (hMG) are common gonadotropins used for superovulation and appropriate concentration of them might be necessary. However, there is still no systematic study on the similarities and differences of DNA methylation alterations induced by superovulation with different dosage of FSH/hMG at the single cell levels. Methods: In the current study, the different doses of FSH/hMG combined with (add hcg full name here) hCG were used in C57BL/6 strain female mice to get experimental group oocytes while naturally matured oocytes and superovulated oocytes with only hCG were respectively set as controls. Single-cell level DNA methylation sequencing was carried out to all the mature oocytes to investigate the effect of superovulation with different dosage of FSH/hMG on DNA methylation during oocyte maturation. Results: In this study, we found that the hypo differentially methylated regions (DMRs) number of FSH/hMG 200 IU group both increased significantly although the whole genome methylation pattern of mature oocytes derived from superovulation were unaffected as compared with the control matured oocytes, suggesting that high dosage of FSH/hMG might lead to increased genomic methylation instability. At the same time, some of the DMRs annotated genes related to oocyte quality and embryonic development potential were disturbed in all dosage of FSH/hMG group (including Twist2, Cdk1, Ntrk2, Slc22a4, Sstr2, Wnt2, Cntnap5a, Gm2087, Slc16a7, and so on) as well as the certain ones (containing Abcc3, Zfp532, Atp1a1, Zfp804a, and so on) were impaired after high dosage of gonadotropins. We speculated that the effect of superovulation on DNA methylation during oocyte maturation might be dose-dependent to a certain extent, and such effect might affect oocyte quality and embryonic development competency. Conclusions: The current study firstly analyzed the differential impacts of different doses of gonadotropins on single-cell level DNA methylation of mouse oocytes, indicating a dose-dependent effect of superovulation with FSH/hMG and hCG on the epigenetic variations in mouse oocytes. Furthermore, some important genes that play a critical role in oocyte quality and embryo development were indicated to form the possible molecular basis of the corresponding regulation. This study laid a foundation for evaluating the safety of superovulation and highlighted the need for more and further research into ART.
2020-06-05 | GSE136730 | GEO
Project description:Vitrification is a method for long-term biological sample cryopreservation without causing intra- and extra-cellular ice formation. We recently established a novel closed vitrification system to cryopreserve mouse ovarian follicles. Using the 3D alginate hydrogel encapsulated in vitro follicle growth (eIVFG) method, we have demonstrated that compared to freshly-harvested follicles, vitrified follicles have normal follicle and oocyte reproductive outcomes. Our recent study further demonstrated the faithful preservation of molecular signatures of follicle-stimulating hormone (FSH)-stimulated follicle maturation in vitrified follicles. However, it is unknown whether ovulation, another crucial gonadotropin-dependent follicular event, and involved ovulatory gene regulatory pathways are well conserved in vitrified follicles. Fresh and vitrified follicles grown for 8 days by eIVFG were collected at 0, 1, 4 and 8-hour post human chorionic gonadotropin (hCG) treatment for the single-follicle RNA sequencing. Principal component analysis (PCA) and Pearson’s correlation analysis revealed that vitrified follicles have similar transcriptomic profiles to fresh follicles. Furthermore, the expression of several genes essential for ovulation were comparable between vitrified and fresh follicles. In summary, these results demonstrate that our closed vitrification system preserves follicular transcriptomic dynamics during ex vivo ovulation. Together with our previous findings that vitrification preserves FSH-stimulated follicle maturation, the integration of vitrification of immature follicles and eIVFG has a great potential to serve as an additional fertilization preservation approach for young cancer patients and endangerers species. Moreover, follicle vitrification enables a high-content ovarian follicle biobank, which can greatly improve the throughput of eIVFG for studying ovulation biology, anovulatory disease, toxicology, and novel contraceptive drug development targeting ovulation.
2023-09-20 | GSE227091 | GEO