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

Abstract

Study question: Can RNA sequencing of human cumulus cells (CC) reveal molecular pathways involved in the physiology of reproductive aging?

Study finding: Senescent but not young CC activate gene pathways associated with hypoxia and oxidative stress.

What is known already: 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.

Study design, samples/materials, methods: 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.

Main results and the role of chance: 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.

Limitations, reasons for caution: 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.

Wider implications of the findings: 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.

Large scale data: GEO accession number: GSE81579 STUDY FUNDING AND COMPETING INTERESTS: Funded in part by EMD Serono Grant for Fertility Innovation (GFI).

Keywords: RNA sequencing; WGCNA; cumulus cells transcriptome; oocyte senescence.

Figure 1

Expression levels of the 45 differentially expressed genes. (A) A heatmap comparing counts per million (CPM) count of each gene for each sample. The intensity of the pseudocolors reflects gene expression levels (red indicates high CPM count, navy indicates low CPM count). Hierarchical clustering of the DE genes shows separation between older and younger groups (B) Plot of differentially expressed genes (left side) comparing samples from younger and older individuals. Differential expression is represented as a fold-change, calculated as the ratio between the CPM of older vs. younger: a positive value indicates a higher expression in the older and consequent lower expression in younger, while a negative value indicates a higher expression in younger as opposed to older. The asterisks indicate genes validated with qPCR (Fig. S2).

Figure 2

Weighted gene co-expression network analysis (WGCNA) and immune fluorescent staining of NOS2 (A) WGCNA analysis revealing co-expressed genes (or modules), which are compared to differentially expressed (DE) genes using a Venn diagram. Modules overlapping with DE genes (modules 0, 1 and 2) are shown. Module 0 displays 28 genes, Module 1 displays 9 genes and Module 2 displays 5 genes overlapping with the list of differentially expressed transcripts. The squares list the overlapping genes. (B) Fluorescent immunostaining of CCs from patients of different age. Nuclei are labeled in blue (DAPI staining). The red signal represents NOS2 protein. (C) Relative NOS2 protein expression in older and younger groups (see Methods). The asterisk indicates P-value significance of <0.05; bars represent standard deviation of the mean.