(66) Impacto da suplementação de melatonina pré-IVM no metabolismo lipídico do oócito e no desenvolvimento do embrião

Laryssa Ketelyn L. Pimenta¹ ; Nayara R. Kussano² ; José Eduardo V. Chaves¹ ; Hallya Beatriz S. Amaral³ ; Maurício M. Franco² ; Margot A. N. Dode^{2, 3} ; José Felipe W. Sprícigo¹

1. University Federal of Goiás, Goiânia, Goiás, Brasil

2. Embrapa Genetic Resources and Biotechnology, Animal Reproduction Laboratory, Brasília, DF, Brasil

3. University of Brasília, Brasília, DF, Brasil

Abstract Text:

In vitro maturation (IVM) can cause asynchrony between nuclear and cytoplasmic maturation, compromising oocyte quality and ultimately embryo development. Pre-maturation may help address this issue. Additionally, lipid accumulation during IVM negatively impacts embryos. Given its antioxidant activity and role in lipid metabolism, melatonin could be a potential solution. Despite the known benefits of adding melatonin in IVM, its effect during pre-IVM, remain unexplored.  We hypothesized that melatonin supplementation during pre-IVM could regulate lipid metabolism, reducing lipid accumulation during IVM and improving embryonic development. To test this, cumulus-oocyte complexes (COCs) were randomly distributed into three experimental groups: (1) Control: COCs IVM for 24 h; (2) Pre-IVM control: COCs pre-matured for 6 h, followed by 24 h of IVM; and (3) Pre-IVM+MTn: COCs pre-matured with melatonin for 6 h, followed by 24 h of IVM. COCs from all groups were evaluated at 0 h for immature control (IC) and 24 h for matured control (MC); after 6 h of pre-IVM and after 24 h of IVM for the groups without (PMIC and PMMC) and with melatonin supplementation (PMMI and PMMM). Oocytes were assessed for nuclear maturation (Hoechst 33342), lipid content (BODIPY), and mitochondrial activity (MitoTracker Deep Red FM) using confocal microscopy. Lipid metabolism-related gene expression was quantified by qPCR in cumulus cells (PPARγ, FABP3) and oocytes (PPARγ, CPT1A, ACSS2, FABP3, PLIN2). After maturation COCs were co-incubated with sperm for 18 h, and zygotes were culture in vitro (IVC) for seven days. Cleavage rate was evaluated on day 2 (D2), and blastocyst rate on day 7 (D7). All culture steps (IVM, IVF, and IVC) were conducted at 38.5°C under 5% O₂ and 5% CO₂. Statistical analyses were performed by ANOVA or t-test for parametric data and by Kruskal-Wallis or Mann-Whitney tests for nonparametric data. Embryonic development was developed by the chi-square test. Differences were considered significant at p ≤ 0.05. Pre-IVM treatment, whether in the absence or presence of melatonin, did not interfere with the oocytes’ ability to complete nuclear maturation, since the majority of oocytes were at MII at 24 h of maturation (MC:17/18, 94.4%; PMMC: 9/9, 100%;  PMMM: 12/12, 100%). The presence of melatonin during pre-IVM reduced oocyte lipid content, with the PMMI group showing the lowest level by the end of maturation (p ≤ 0.05) Meanwhile, although mitochondrial activity increased (p ≤ 0.05) in all groups during IVM, it reached similar levels (p ≤ 0.05) across groups at the end of maturation. Regarding to gene expression, only FABP3 showed decreased transcript levels in cumulus cells of the pre-IVM groups (PMMC and PMMM) compared to the MC group (p < 0.05), regardless of melatonin exposure, while no differences were observed in oocyte gene expression among treatments. The cleavage rate was higher (p ≤ 0.05) in the pre-IVM control (442/557; 79.4%) and pre-IVM+MTn (451/601; 75%) groups compared to the control group (392/594; 66.0%). This difference was maintained, as on D7, blastocyst rates remained higher in the pre-IVM (200/557; 35.9%) and pre-IVM+MTn (185/601; 30.8%) groups than in the control group (153/594; 25.8%). It is concluded that the use of melatonin in pre-IVM can reduce the level of lipid droplets stored at the end of IVM. However, this effect was not mediated by the genes investigated. Furthermore, melatonin supplementation did not potentiate the beneficial effect of pre-IVM regarding embryo development.