Effects of chemical in vitro activation versus fragmentation on human ovarian tissue and follicle growth in culture
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PROVIDER: PRJNA982873 | ENA |
REPOSITORIES: ENA
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Project description:Abstract STUDY QUESTION What is the effect of the chemical in vitro activation (cIVA) protocol compared with fragmentation only (Frag, also known as mechanical IVA) on gene expression, follicle activation, and growth in human ovarian tissue in vitro? SUMMARY ANSWER Although histological assessment shows that cIVA significantly increases follicle survival and growth compared to Frag, both protocols stimulate extensive and nearly identical transcriptomic changes in cultured tissue compared to freshly collected ovarian tissue, including marked changes in energy metabolism and inflammatory responses. WHAT IS KNOWN ALREADY Treatments based on chemical in vitro activation of the phosphatase and tensin homolog (PTEN)- phosphatidylinositol 3-kinase (PI3K) pathway in ovarian tissue followed by auto-transplantation have been administered to patients with refractory premature ovarian insufficiency (POI) and resulted in live births. However, comparable effects with mere tissue fragmentation have been shown, questioning the added value of chemical stimulation that could potentially activate oncogenic responses. STUDY DESIGN, SIZE, DURATION Fifty-nine ovarian cortical biopsies were obtained from consenting women undergoing elective caesarean section (C-section). The samples were fragmented for culture studies. Half of the fragments were exposed to bpV (HOpic) + 740Y-P (Frag + cIVA group) during the first 24 h of culture, while the other half were cultured with medium only (Frag group). Subsequently, both groups were cultured with medium only for an additional 6 days. Tissue and media samples were collected for histological, transcriptomic, steroid hormone and cytokine/chemokine analyses at various time points. PARTICIPANTS/MATERIALS, SETTING, METHODS Effects on follicles were evaluated by counting and scoring serial sections stained with hematoxylin and eosin before and after the 7-day culture. Follicle function was assessed by quantification of steroids by ultra-performance liquid chromatography tandem-mass spectrometry at different time points. Cytokines and chemokines were measured by multiplex assay. Transcriptomic effects were measured by RNA-sequencing (RNA-seq) of the tissue after the initial 24 h culture. Selected differentially expressed genes (DEGs) were validated by quantitative PCR and immunofluorescence in cultured ovarian tissue as well as in KGN cell (human ovarian granulosa-like tumor cell line) culture experiments. MAIN RESULTS AND THE ROLE OF CHANCE Compared to the Frag group, the Frag + cIVA group exhibited a significantly higher follicle survival rate, increased numbers of secondary follicles, and larger follicle sizes. Additionally, the tissue in Frag + cIVA group produced less dehydroepiandrosterone compared to Frag. Cytokine measurement showed a strong inflammatory response at the start of the culture in both groups. The RNA-seq data revealed modest differences between the Frag + cIVA and Frag groups, with only 164 DEGs identified using a relaxed cut-off of false discovery rate (FDR)<0.1. Apart from the expected PI3K-protein kinase B (Akt) pathway, cIVA also regulated pathways related to hypoxia, cytokines, and inflammation. In comparison to freshly collected ovarian tissue, gene expression in general was markedly affected in both Frag + cIVA and Frag groups, with a total of 3,119 and 2,900 DEGs identified (FDR < 0.001), respectively. The top enriched gene sets in both groups included several pathways known to modulate follicle growth such as mammalian target of rapamycin (mTOR)C1 signaling. Significant changes compared to fresh tissue were also observed in the expression of steroidogenesis enzymes and classical granulosa cell markers in both groups. Intriguingly, we discovered a profound upregulation of genes related to glycolysis and its upstream regulator in both Frag and Frag + cIVA groups, and these changes were further boosted by the cIVA treatment. Cell culture experiments confirmed glycolysis-related genes as direct targets of the cIVA drugs. In conclusion, cIVA enhances follicle growth, as expected, but the mechanisms may be more complex than PI3K-Akt-mTOR alone, and the impact on function and quality of the follicles after the culture period remains an open question. LARGE SCALE DATA Data was deposited in GEO data base, accession number GSE234765. Code for sequencing analysis can be found in https://github.com/tialiv/IVA_project. LIMITATIONS, REASONS FOR CAUTION Similar to the published IVA protocols, the first steps in our study were performed in an in vitro culture model where the ovarian tissue was isolated from the regulation of hypothalamic-pituitary-ovarian axis. Further in vivo experiments will be needed, for example in xeno-transplantation models, to explore the long-term impacts of the discovered effects. The tissue collected from patients undergoing C-section may not be comparable to tissue of patients with POI. WIDER IMPLICATIONS OF THE FINDINGS The general impact of fragmentation and short (24 hours) in vitro culture on gene expression in ovarian tissue far exceeded the effects of cIVA. Yet, follicle growth was stimulated by cIVA, which may suggest effects on specific cell populations that may be diluted in bulk RNA-seq. Nevertheless, we confirmed the impact of cIVA on glycolysis using a cell culture model, suggesting impacts on cellular signaling beyond the PI3K pathway. The profound changes in inflammation and glycolysis following fragmentation and culture could contribute to follicle activation and loss in ovarian tissue culture, as well as in clinical applications, such as fertility preservation by ovarian tissue auto-transplantation.
2024-05-24 | GSE234765 | GEO
Project description:Human hair follicles from normal areas of the scalp were disassociated to single cells, sorted and tested by microarrray To compare the expression of human CD200+ CD49+ hair follicle keratinocytes versus CD200-CD49+ keratinocytes
2011-01-12 | GSE21567 | GEO
Project description:Ovarian follicle selection plays an important role in the reproduction of sexually mature hens, and this process can directly affect the growth and development of follicles until the final ovulation, thus affecting laying performance and fecundity of hens. In the laying hen ovary, one follicle from a cohort of 8-13 follicles of 6-8 mm in diameter is selected daily to enter the preovulatory hierarchy. In this study, we globally compared the proteomes of chicken ovarian follicles before and after follicle selection. A total of 5883 proteins were identified in the proteomes of chicken 6-8 mm prehierarchical follicles and 12-15 mm hierarchical follicles. 259 proteins are differentially expressed in 12-15 mm hierarchical follicle compared with prehierarchical follicle, of which 175 proteins are up-regulated and 84 proteins are down-regulated. The Gene Ontology enrichment of differentially expressed proteins revealed enriched GO terms for peptidase activity, acrosin binding for their molecular function and in the process of negative regulation of peptidase activity, and regulation of fertilization. The KEGG pathway analysis indicated that differentially expressed proteins were enriched for ribosome, lysine degradation, and endocytosis pathways. Nine differentially expressed proteins including vitellogenin-1 were validated with Parallel Reaction Monitoring (PRM) analysis, and their functions were discussed. This study provided a global proteomic view of the development of chicken ovarian follicles, which will serve as a foundation for understanding the molecular signatures and pathways of follicle selection in hens.
2021-09-08 | PXD011470 | Pride
Project description:We use DSN normalized RNA-seq to transcriptionally profile FACS sorted 16C ovarian follicle cells. These data provide insights into the developmental control of gene expression programmed gene amplificaton. Follicle cells were isolated from whole ovaries by trypsinization and filtering and stained with Hoescht. 16C follicle cells were isolated by FACS sorting based on DNA content (Hoescht). RNA was extracted with TRIzol reagent and 100ng of total RNA and used to generate a total library. This library was then subjected to DSN normalization prior to Illumina based sequencing.
2011-05-27 | E-GEOD-29526 | biostudies-arrayexpress
Project description:Primary outcome(s): Comparison of the gene expression profile in hair follicle between cancer patients and healthy volunteer
| 2628246 | ecrin-mdr-crc
Project description:Thecal tissue forms a layer around the follicle just prior to antral stage and grows with the follicle (containing an oocyte) as it matures. The innermost component (theca interna) supplies hormones and other factors necessary to the growth and development of the granulosa and oocyte. Most follicles regress and die (become atretic) at the antral stage, and this process as well as development of the follicle are undoubtedly influenced by the theca. Transcriptional changes in ovarian theca interna at the global level were examined by microarray during atresia and development to improve our understanding of the mechanisms involved. Four groups of bovine ovarian follicles were selected for analysis . Follicle size, type and array number for each group are, small (3-5 mm) healthy rounded (n=5), healthy columnar (n=5), atretic(n=5) and large healthy (>9mm), (n=4). For each group, the RNA from the theca interna of a single follicle was used to hybridise an array.
2014-05-20 | E-GEOD-49505 | biostudies-arrayexpress
Project description:Primordial follicle assembly is the process by which ovarian primordial follicles are formed. During follicle assembly oocyte nests break down and a layer of pre-granulosa cells surrounds individual oocytes to form primordial follicles. The pool of primordial follicles formed is the source of oocytes for ovulation during a femaleM-bM-^@M-^Ys reproductive life. Complex networks of cellular signaling and gene expression are essential for any biological process. A systems biology experimental approach provides a global view of these gene relationships in a particular developmental process. The current study utilized a systems approach to detect all genes that are differentially expressed in response to seven different growth factor and hormone treatments known to influence primordial follicle assembly in a neonatal rat ovary culture system. One novel growth factor, basic fibroblast growth factor (FGF2), was experimentally determined to inhibit follicle assembly. The different growth factor and hormone treatments were all found to affect the same physiological pathways, but each treatment affected a unique set of differentially expressed genes (signature gene set). A gene bionetwork analysis identified gene modules of coordinately expressed interconnected genes and it was found that different gene modules appear to accomplish distinct tasks during primordial follicle assembly. Unique gene networks were identified for a number of the modules and signature gene sets. Predictions of physiological pathways important to follicle assembly were validated using ovary culture experiments in which ERK1/2 (MAPK1) activity was increased. A number of the highly interconnected genes in these gene networks have previously been linked to primary ovarian insufficiency (POI) and polycystic ovarian disease syndrome (PCOS). Observations have identified novel factors and gene networks that regulate primordial follicle assembly. This systems approach has helped elucidate the molecular control of primordial follicle assembly and provided potential therapeutic targets for the treatment of ovarian disease. We used microarrays to determine genes expressed differentially between control and P0 ovaries treated with 7 growth factors: AMH, CTGF, estradiol (E2), Activin-a, FGF2, progesterone (P4), and TNFa. RNA samples from 7 control samples are compared to 3 growth factor treated ovary samples for each AMH (human Anti-MM-CM-<lerian hormone), CTGF (connective tissue growth factor ), estradiol (E2), TNF (tumor necrosis factor ), FGF2 (fibroblast growth factor 2), Inhba (inhibin, beta A), and 2 samples for progesterone (P4).
2013-01-15 | E-GEOD-43464 | biostudies-arrayexpress
Project description:Primordial follicle assembly is the process by which ovarian primordial follicles are formed. During follicle assembly oocyte nests break down and a layer of pre-granulosa cells surrounds individual oocytes to form primordial follicles. The pool of primordial follicles formed is the source of oocytes for ovulation during a female’s reproductive life. Complex networks of cellular signaling and gene expression are essential for any biological process. A systems biology experimental approach provides a global view of these gene relationships in a particular developmental process. The current study utilized a systems approach to detect all genes that are differentially expressed in response to seven different growth factor and hormone treatments known to influence primordial follicle assembly in a neonatal rat ovary culture system. One novel growth factor, basic fibroblast growth factor (FGF2), was experimentally determined to inhibit follicle assembly. The different growth factor and hormone treatments were all found to affect the same physiological pathways, but each treatment affected a unique set of differentially expressed genes (signature gene set). A gene bionetwork analysis identified gene modules of coordinately expressed interconnected genes and it was found that different gene modules appear to accomplish distinct tasks during primordial follicle assembly. Unique gene networks were identified for a number of the modules and signature gene sets. Predictions of physiological pathways important to follicle assembly were validated using ovary culture experiments in which ERK1/2 (MAPK1) activity was increased. A number of the highly interconnected genes in these gene networks have previously been linked to primary ovarian insufficiency (POI) and polycystic ovarian disease syndrome (PCOS). Observations have identified novel factors and gene networks that regulate primordial follicle assembly. This systems approach has helped elucidate the molecular control of primordial follicle assembly and provided potential therapeutic targets for the treatment of ovarian disease. We used microarrays to determine genes expressed differentially between control and P0 ovaries treated with 7 growth factors: AMH, CTGF, estradiol (E2), Activin-a, FGF2, progesterone (P4), and TNFa.
2013-01-15 | GSE43464 | GEO
Project description:Thecal tissue forms a layer around the follicle just prior to antral stage and grows with the follicle (containing an oocyte) as it matures. The innermost component (theca interna) supplies hormones and other factors necessary to the growth and development of the granulosa and oocyte. Most follicles regress and die (become atretic) at the antral stage, and this process as well as development of the follicle are undoubtedly influenced by the theca. Transcriptional changes in ovarian theca interna at the global level were examined by microarray during atresia and development to improve our understanding of the mechanisms involved.
2014-05-20 | GSE49505 | GEO
Project description:Study questionCan cultured follicles model the ovarian cycle, including follicular- and luteal-phase hormone synthesis patterns and ovulation?Summary answerUnder gonadotrophin stimulation, murine follicles grown in an encapsulated three-dimensional system ovulate in vitro and murine and human follicle hormone synthesis mimics follicular and luteal phases expected in vivo.What is known alreadyStudies of the human ovary and follicle function are limited by the availability of human tissue and lack of in vitro models. We developed an encapsulated in vitro follicle growth (eIVFG) culture system, which preserves 3D follicular structure. Thus far, the alginate system has supported the culture of follicles from mice, dog, rhesus macaque, baboon and human. These studies have shown that cultured follicles synthesize steroid hormones similar to those observed during the follicular phase in vivo.Study design, size, durationCultured murine follicles were treated with human chorionic gonadotrophin (hCG) and epidermal growth factor (EGF) and either assayed for luteinization or removed from alginate beads and assayed for ovulation. Human follicles were also cultured, treated with follicle-stimulating hormone (FSH), hCG and EGF to mimic gonadotrophin changes throughout the ovarian cycle, and culture medium was assayed for hormone production.Participants/materials, setting, methodsMurine and human follicles were cultured in alginate hydrogel and hormone production [17β-estradiol, progesterone, inhibin A, inhibin B, activin A and anti-Müllerian hormone (AMH)] was quantified in medium by enzyme-linked immuno assay (ELISA). Human ovarian tissue was acquired from females between 6 and 34 years of age with a cancer diagnosis. These participants were undergoing ovarian tissue cryopreservation at National Physicians Cooperative sites as part of the Oncofertility Consortium.Main results and the role of chanceWhen grown in this system, 96% of mouse follicles ovulated in response to hCG and released meiotically competent eggs. Ovulated follicles recapitulated transcriptional, morphologic and hormone synthesis patterns post-luteinizing hormone (LH/hCG). In addition to rodent follicles, individual human follicles secreted steroid and peptide hormones that mimicked the patterns of serum hormones observed during the menstrual cycle.Limitations, reasons for cautionThis was a descriptive study of an in vitro model of ovulation and the ovarian hormone cycle. The ovulation studies were limited to murine tissue and further studies are needed to optimize conditions using other species.Wider implications of the findingsThe eIVFG system reliably phenocopies the in vivo ovarian cycle and provides a new tool to study human follicle biology and the influence of cycling female hormones on other tissue systems in vitro.Study funding/competing interestsThis work was supported by NIH U54 HD041857, NIH U54 HD076188, NIH UH2 E5022920, NIH UH3 TR001207 and F30 AG040916 (R.M.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
| S-EPMC4447886 | biostudies-literature