<em>Effect of cumulin on de-differentiation of human post-ovulatory cumulus cells</em> — ASN Events
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Effect of cumulin on de-differentiation of human post-ovulatory cumulus cells (#498)

Ananya Vuyyuru 1 , Amelia Scobie 1 , Nicola J Byers 1 , Emily R Frost 1 , Robert B Gilchrist 1
  1. Fertility & Research Centre, Discipline of Women's Health, School of Clinical Medicine, University of New South Wales, Sydney, New South Wales, Australia

Background: Within large antral ovarian follicles, there is a tightly regulated signalling balance to control the specification and differentiation of mural granulosa cells and the oocyte-adjacent cumulus cells. Oocyte-secreted factors (OSFs) such as growth differentiation factor-9 (GDF9) and bone morphogenetic protein 15 (BMP15) play key roles opposing FSH to establish and maintain the cumulus cell phenotype. However, it remains unknown whether OSFs can de-differentiate and restore the preovulatory cumulus phenotype in post-ovulatory cells collected from human ICSI stimulation cycles.

Methods: Human post-ovulatory cumulus cells were obtained from consented patients undergoing an ICSI cycle at the Fertility & Research Centre, Royal Hospital for Women, Randwick. Cells were cultured for 48 hr, before treatment ± FSH (50 mIU/mL) and ± pro-cumulin (50 ng/mL), a recombinant GDF9:BMP15 heterodimer. A panel of gene markers for cumulus expansion, steroidogenesis/luteinisation, and granulosa identity were assessed using qPCR (n=6) and immunocytochemistry was performed for FOXL2 (a granulosa cell marker) and PTX3 (a cumulus expansion marker).

Results: FSH-treated cumulus cells showed an elevation in luteinisation and steroidogenesis genes when compared with cells treated with pro-cumulin (LHCGR, 4.8-fold, p= 0.0269; STAR, 8.2-fold, p<0.0001; CYP11A1, 3.0-fold, p=0.0200), indicating that FSH treatment drives a mural granulosa cell phenotype. In contrast, pro-cumulin treatment stimulated a cumulus cell expansion gene (HAS2, 5.5-fold, p=0.0264), along with other key markers of the cumulus cell phenotype (AR, 2.5-fold, p=0.0041; SFRP4, 4.1-fold, p<0.0001). Immunofluorescence staining revealed expression of FOXL2 and PTX3 in all four treatment groups, with quantitative analysis underway to confirm alterations in protein expression.

Conclusions: This study demonstrates that human post-ovulatory cumulus cells exhibit differentiation plasticity at the gene level and that pro-cumulin re-engages cumulus-specific transcripts and attenuates luteinisation markers, generating a molecular transcriptomic signature more aligned with a pre-ovulatory cumulus cell phenotype.