Oral Presentation ESA-SRB-NZSE-CaSR 2026 in conjunction with ENSA

Can denuded ART oocytes destined for waste be rescued A pipeline for re-establishing somatic cell support (143596)

Nicola Byers 1 2 , Amelia Scobie 2 , Benjamin S Wu 3 , Jared M Campbell 1 , Lindsay E Wu 3 , Emily R Frost 2 , Ewa M Goldys 1 , Robert B Gilchrist 2
  1. School of Biomedical Engineering, University of New South Wales, Sydney, NSW, Australia
  2. Fertility & Research Centre, Discipline of Women’s Health, School of Clinical Medicine, University of New South Wales, Sydney, NSW, Australia
  3. School of Biomedical Sciences, University of New South Wales, Sydney, NSW, Australia

Background

Approximately 5–15% of ART oocytes for ICSI are discarded due to incomplete maturation after hormonal triggers. Because these oocytes are denuded for assessment prior to insemination, they prematurely lose critical gap-junctional signalling from the surrounding cumulus cells (CCs), compromising developmental competence. There is currently no effective rescue mechanism to mature and fertilise these oocytes; therefore, this study aims to establish a reaggregation protocol to restore cumulus-oocyte complexes (COCs) in immature oocytes.

Methods

Swiss mice were used to establish a CC-oocyte reaggregation protocol. Transzonal projection (TZP) phalloidin immunofluorescence after reaggregation, calcein dye to assess functionality of re-formed TZPs, and IVM and IVF were used to assess reaggregation efficiency. Reaggregation of post-ovulatory human CCs and discarded oocytes was attempted using fresh CCs or cells pre-cultured for 72h in an effort to revert CCs to a pre-ovulatory-like state, as assessed in qRT-PCR.

Results

Our protocol led to the re-formation of murine COCs after 48h reaggregation. Staining showed that TZPs were present (all COCs phalloidin positive), and appearance of calcein dye in unstained oocytes reaggregated with stained CCs indicated reformation of functional TZPs (integrated density 401982 vs. 68293 [non-stained control]). IVM and subsequent IVF showed reaggregated oocytes could successfully mature and fertilise, with 37.0% developed to the blastocyst stage compared to 4.8% non-reaggregated control. In human, reaggregation was unsuccessful using fresh or pre-cultured CCs, although qPCR data suggested that prolonged culture of hCCs may de-differentiate CCs to a state more capable of reaggregation, with decreased STAR and PTX3 (15-fold, p<0.0001 and 11-fold, p<0.001]), and increased FSHR (17-fold, p<0.0001).

Conclusion

To our knowledge, this is the first protocol that fully reforms the COCs of denuded mouse GV oocytes. This is the first step in establishing a protocol that rescues denuded ART oocytes, which would give patients more eggs and thus improve fertility prospects.