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Transcriptome analysis of human cumulus cells reveals hypoxia as the main determinant of follicular senescence.

ABSTRACT: Can RNA sequencing of human cumulus cells (CC) reveal molecular pathways involved in the physiology of reproductive aging?Senescent but not young CC activate gene pathways associated with hypoxia and oxidative stress.Shifts in socioeconomic norms are resulting in larger numbers of women postponing childbearing. The reproductive potential is sharply decreased with aging, and the reasons are poorly understood. Since CCs play an integral role in oocyte maturation and direct access to human oocytes is limited, we used whole transcriptome analysis of these somatic cells to gain insights into the molecular mechanisms playing a role in follicular senescence.Twenty CC samples (from a total of 15 patients) were obtained from oocytes of either male factor or egg donor patients. RNA sequencing and bioinformatic tools were used to identify differentially expressed genes between CCs from seven aged and eight young patients (<35 (years old) y.o. vs >40 y.o.). Quantitative-PCR and immunoflourescent staining were used for validation.RNA sequencing identified 11 572 genes expressed in CC of both age cohorts, 45 of which were differentially expressed. In CC collected from patients >40 y.o., genes involved in the hypoxia stress response (NOS2, RORA and NR4A3), vasculature development (NR2F2, PTHLH), glycolysis (RALGAPA2 and TBC1D4) and cAMP turnover (PDE4D) were significantly overexpressed when compared with CC of patients younger than 35 y.o.This study focused almost exclusively on assessing the genetic differences in CC transcriptome between young and older women. These genetic findings were not fully correlated with embryonic development and clinical outcome.Our data provide a new hypothesis-follicular hypoxia-as the main mechanism leading to ovarian follicular senescence and suggest a link between cumulus cell aging and oocyte quality decay. If specific molecular findings of hypoxia would be confirmed also in oocytes, genetic platforms could screen CC for hypoxic damage and identify healthier oocytes. Protocols of ovarian stimulation in older patients could also be adjusted to diminish oocyte exposure time to hypoxic follicles.GEO accession number: GSE81579 STUDY FUNDING AND COMPETING INTERESTS: Funded in part by EMD Serono Grant for Fertility Innovation (GFI).

SUBMITTER: Molinari E

PROVIDER: S-EPMC4986421 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Transcriptome analysis of human cumulus cells reveals hypoxia as the main determinant of follicular senescence.

Molinari E E Bar H H Pyle A M AM Patrizio P P

Molecular human reproduction 20160606 8

<h4>Study question</h4>Can RNA sequencing of human cumulus cells (CC) reveal molecular pathways involved in the physiology of reproductive aging?<h4>Study finding</h4>Senescent but not young CC activate gene pathways associated with hypoxia and oxidative stress.<h4>What is known already</h4>Shifts in socioeconomic norms are resulting in larger numbers of women postponing childbearing. The reproductive potential is sharply decreased with aging, and the reasons are poorly understood. Since CCs pla ...[more]

PMID: 27268410

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Project description:Purpose: The goal of this study was to identify differentially expressed genes and pathways between the cumulus of compact/unstimulated cumulus-oocyte-complex (COC) and the cumulus of expanded/stimulated COC. Methods: mRNA profiles of Compact/unstimulated cumulus cells (CCs) from germinal vesicle (GV) COC obtained from two patients undergoing unstimulated IVM procedure and expanded/stimulated CCs from metaphase 2 (MII) COC obtained from three patients undergoing IVF/ICSI were generated by deep sequencing using Illumina HiSeq 2000. The sequence reads that passed quality filters were mapped to the human genome (hg19) using Tophat software. Differential expression analysis was done using DESeq bioconductor package. qRT–PCR validation was performed using SYBR Green assays. Results: A total of 40-80 million sequence reads per sample were mapped to the human genome (hg19). A total of 1746 differentially expressed genes between compact and expanded CCs with fold change > 2, and adjusted p value < 0.05 were identified. Gene ontology analysis of differentially expressed genes revealed a number of cellular processes regulated during the periovulatory interval including cellular movement, inflammatory response, immune cell trafficking, tissue development, lipid metabolism, tissue morphology, DNA replication and cell cycle. A total of 116 of the differentially expressed genes were annotated as long non-coding RNAs, 10 of them coded from introns of genes known to be involved in granulosa cell processes suggesting that unique non coding RNA transcripts may contribute to the regulation of cumulus expansion and oocyte maturation. Results were validated using qRT-PCR. Conclusions: Using global transcriptome sequencing we identified new important genes and non coding RNAs involved in COC maturation and cumulus expansion, which may contribute to improve the process of in vitro maturation of immature oocytes utilized in IVM cycles mRNA profiles of Compact/unstimulated cumulus cells (CCs) from germinal vesicle (GV) COC obtained from two patients undergoing unstimulated IVM procedure and expanded/stimulated CCs from metaphase 2 (MII) COC obtained from three patients undergoing IVF/ICSI were generated by deep sequencing using Illumina HiSeq 2000.

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Transcriptome analysis of human cumulus cells reveals hypoxia as the main determinant of follicular senescence.

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Transcriptome analysis of human cumulus cells reveals hypoxia as the main determinant of follicular senescence.

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