JBRA Assisted Reproduction 2025;29(Suppl.2 SBRA 2025):236
Poster Presentation
29^th Annual Congress of the SBRA. São Paulo/SP - Brazil, 2025
doi: 10.5935/1518-0557.20263845

P-224. The effects of post-ovulatory aging in female reproductive health: how does it impact the morphology and function of cumulus-oocyte complexes and oocytes?

Veronica Louzada Reis¹, Fabiola Freitas Paula-Lopes¹

¹ Universidade Federal de São Paulo - Diadema – SP- Brasil

Objective: Post-ovulatory aging (POA) is responsible to generate a profound effect on female fertility, occurring when fertilization fails to take place within the optimal time frame after the metaphase II oocyte ovulation. POA manifests in both in vitro and in vivo environments, adversely affecting oocyte quality and competence, leading to abnormal fertilization rates and embryo developmental competence. Nevertheless, the effects of POA exposure on intact cumulus-oocyte complexes (COCs) compared to denuded oocytes remain poorly understood. Therefore, this study employed a bovine model to investigate cellular and morphological responses to POA, evaluating both intact COCs and denuded oocytes.
Methods: Bovine ovaries obtained from a slaughterhouse were processed for COC isolation and allocated into three groups: Immature (0 h in vitro maturation — IVM), Mature (22 h IVM), and Aged (40 h prolonged IVM). Oocytes from each treatment were separated into two subgroups: intact COCs and denuded oocytes, obtained by removing cumulus cells. These samples were used to assess mitochondrial activity using MitoTracker Red CMXROS (Experiment 1; N=3 replicates) and intracellular reactive oxygen species (ROS) levels using CellROX Green (Experiment 2; N=4 replicates). In addition, TUNEL assay was performed to investigate apoptosis (Experiment 3; N=5 replicates for denuded oocytes and 3 replicates for intact COCs). In Mature and Aged denuded oocytes, meiotic progression was analyzed using Hoechst 33342 staining (Experiment 4; N=5 replicates). For morphological assessments, denuded oocytes were examined to measure zona pellucida thickness, perivitelline space area, and cytoplasmic volume (Experiments 5–7; N=3 replicates), whereas cumulus cells dispersion was evaluated in intact COCs (Experiment 8; N=4 replicates). Sperm attachment to the zona pellucida was measured in Mature and Aged denuded oocytes one hour post-in vitro fertilization (IVF) through Hoechst 33342 staining (Experiment 9; N=3 replicates). Fluorescence analyses were conducted using a fluorescence microscope (Zeiss Axio Imager A2) and Zen Pro sofware, and morphological analyses were performed with an inverted microscope (Zeiss Primo Vert) and Axio Vision software. Image J software was utilized for image quantification. Statistical analyses included Wilcoxon for non-parametric data and ANOVA or T test for parametric data (SAS User's Guide, 1989).
Results: Aged COCs displayed a pronounced reduction in mitochondrial activity compared with all other groups (p<0.0001), whereas no difference was detected between Mature and Aged denuded oocytes. As expected, Immature oocytes exhibited the lowest mitochondrial activity (p<0.0001). ROS levels were markedly higher in Mature and Aged COCs relative to Immature COCs (p<0.0001). In denuded oocytes, POA increased ROS synthesis compared with both Immature (p<0.0001) and Mature (p=0.0002) counterparts. The incidence of apoptosis in denuded oocytes did not differ among the experimental groups. In contrast, cumulus cells from Aged COCs unveiled a significant rise in apoptosis compared with Immature (p=0.0024) and Mature (p=0.0154) COCs. Aging also promoted greater cumulus cell dispersion relative to Mature COCs (p=0.0008). No variation in meiotic progression was observed between Mature and Aged oocytes. Morphological assessment revealed that both Mature and Aged oocytes, when compared with Immature oocytes, presented thicker zona pellucida (p=0.0112), enlarged perivitelline space (p<0.0001), and reduced cytoplasmic volume (p<0.0001), with no differences between the latter two groups. Additionally, sperm binding to the zona pellucida was significantly increased in Aged oocytes in comparison with the Mature treatment (p<0.0001).
Conclusion: In conclusion, POA elicited distinct effects on intact COCs and denuded oocytes. In COCs, POA impaired mitochondrial function, induced cumulus cell apoptosis, and promoted cumulus dispersion. In denuded oocytes, POA was associated with increased ROS production, specific morphological changes and enhanced sperm binding to zona pellucida, although subsequent embryonic competence remains uncertain. These findings highlight that POA affects multiple aspects of oocyte physiology and the fertilization microenvironment, with potential implications for assisted reproduction strategies.