Effects of lactate, super-GDF9 and low oxygen tension during biphasic in vitro maturation on the bioenergetic profiles of mouse cumulus-oocyte-complex

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.

Mouse oocytes restore antral stage partial mechanical denudation in vitro

“…Here, we explored the potential of in vitro and in vivo grown oocytes to restore connectivity with somatic companions prior to maturation, following complete mechanical denudation at late antral follicle stage using our mouse IFC system (Cortvrindt & Smitz, 2002;Saenz-De-Juano et al, 2019). To further extend our reconnection hypothesis to CAPA-IVM, we used our mouse CAPA-IVM system (Romero et al, 2016;Akin et al, 2021Akin et al, , 2022 to test the reconnection capacity of PD-COCs from unprimed 19-21 days old mice during culture. Given their innately lower competence, oocytes from 19-21 days old unprimed mice mimic the low developmental competence of those targeted in human CAPA-IVM (Sanchez et al, 2019).…”