ABSTRACT
In vitro maturation (IVM) is an alternative assisted reproductive technology (ART) with reduced hormone related side-effects and treatment burden compared to conventional IVF. Capacitation (CAPA)-IVM is a biphasic IVM system with improved clinical outcomes compared to standard monophasic IVM. Yet, CAPA-IVM efficiency compared to conventional IVF is still suboptimal in terms of producing utilizable blastocysts. Previously we have shown that CAPA-IVM leads to a precocious increase in cumulus cell (CC) glycolytic activity during cytoplasmic maturation. In the current study, considering the fundamental importance of CCs for oocyte maturation and cumulus-oocyte complex (COC) microenvironment, we further analyzed the bioenergetic profiles of maturing CAPA-IVM COCs. Through a multi-step approach, we (i) explored mitochondrial function of the in vivo and CAPA-IVM matured COCs through real-time metabolic analysis with Seahorse analyzer; and to improve COC metabolism (ii) supplemented the culture media with lactate and/or super-GDF9 (an engineered form of growth differentiation factor 9) and (iii) reduced culture oxygen tension. Our results indicated that the pre-IVM step is delicate and prone to culture related disruptions. Lactate and/or super-GDF9 supplementations failed to eliminate pre-IVM induced stress on COC glucose metabolism and mitochondrial respiration. However, when performing pre-IVM culture under 5% oxygen tension, CAPA-IVM COCs showed a similar bioenergetic profiles compared to in vivo matured counterparts. This is the first study providing real-time metabolic analysis of the COCs from a biphasic IVM system. The currently used analytical approach provides the quantitative measures and the rational basis to further improve IVM culture requirements.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
AUTHORS’ ROLES NA: Designed the experiments, analyzed the data, prepared the manuscript. NA, AH, KB: Performed cultures and in vitro fertilization experiments, collected samples for enzymatic assays. NA, GA: Performed mitochondrial function tests with Seahorse Analyzer and interpreted the data. NA, AH, CM: Performed enzymatic assays. NA, BG: Performed stainings, imaging and image analysis. LVM: Provided supervision on the enzymatic assays. WS, CH: Synthesized the super-GDF9. FK, AM: Provided supervision on the data interpretation. JS, EA: Supervised the project and secured the funding. All authors have read the manuscript and agreed on the final version.
CONFLICT OF INTEREST Authors report no conflict of interest.
FUNDING: This project was funded by the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO) Excellence of Science (EOS, FWO- F.R.S-FNRS; G0F3118N), FWO medium-scale research infrastructure program (I001420N) and the Vrije Universiteit Brussel (OZR). GA is supported by FWO (12B3223N).