136 Progesterone concentration during bovine in vitro maturation might serve as a predictor of oocyte developmental capacity

The inherent quality of the oocyte is one of the limiting factors determining embryo development. Identifying noninvasive cellular or molecular markers that could predict oocyte developmental competence is of significant interest to research and practical applications. This preliminary study aimed to determine the concentration of the steroids oestradiol (E2) and progesterone (P4) during in vitro oocyte maturation (IVM) and fertilisation (IVF) and to correlate hormone levels with IVF outcomes. Cumulus–oocyte complexes (COCs) graded as Class I and II were collected by aspirating follicles (2–6 mm in size) from slaughterhouse-derived ovaries. For IVM, COCs were pooled in groups of 25 to 30 and incubated for 24 h in 250 µL of TCM199, supplemented with 10 IU mL⁻¹ equine chorionic gonadotrophin (eCG) and 5 IU mL⁻¹ human chorionic gonadotrophin (hCG) (Suigonan®, MSD Intervet) without any source of serum. After IVM, expanded COCs were placed in 250 µL of fertilisation medium (Fert-TALP) and fertilised with frozen-thawed sperm from a Holstein bull with proven fertility. Then, 18 to 20 h after IVF, presumptive zygotes were cultured in 500 µL of in vitro culture (IVC) medium based on synthetic oviductal fluid (SOF) enriched with 4 mg mL⁻¹ of fatty acid-free bovine serum albumin covered with 600 µL of mineral oil until Day 8. All in vitro steps were carried out in four-well dishes in a humidified atmosphere at 38.5°C and 5% CO₂ for IVM and IVF and at 38.5°C, 5% CO₂, and 5% O₂ for IVC. Unlike IVC, both IVM and IVF steps were oil-free. Seven IVF cycles were performed. The collected medium after IVM and IVF was centrifuged at 12 000 rpm for 6 min, and the supernatant was frozen at −20°C until hormonal evaluation. The E2 (pg mL⁻¹) and P4 (ng mL⁻¹) concentrations were determined via an established enzyme-linked immunosorbent assay using an ELISA reader (Sunrise™, Tecan). Embryo developmental potential was assessed by determining the number of blastocysts and hatching blastocysts on Day 8 after IVF. Concentrations of E2 and P4 were calculated per COCs during IVM and IVF, and the relationship with in vitro embryo developmental capacity was statistically determined using a bivariate analysis in JMP software (version 16; SAS Institute Inc.). Significant differences were set at P < 0.05. Means for E2/COCs and P4/COCs found during IVM differed (P < 0.05) from that observed during IVF (6.1 ± 1.5 pg mL⁻¹ and 0.37 ± 0.04 ng mL⁻¹ vs. 0.5 ± 0.1 pg mL⁻¹ and 1.2 ± 0.07 ng mL⁻¹, respectively). Bivariate analysis showed that P4/COCs concentrations affected blastocyst rate (P < 0.01) and hatching rate (P < 0.05). One-way ANOVA revealed that P4/COCs with >0.38 ng mL⁻¹ during IVM showed higher (P < 0.05) embryo developmental capacity compared to P4/COCs with < 0.38 ng mL⁻¹ (blastocyst rate: 52.5 ± 4.3% vs. 33.4 ± 1.9%; hatching rate: 30.6 ± 2.7% vs. 11.6 ± 6.4%, respectively). These data show that the concentration of P4 produced by COCs during IVM affects in vitro embryo developmental capacity and warrants further study in understanding the molecular pathways involved in bovine oocyte competence acquisition and as a noninvasive test for developmental potential.