Significance: Four decades have passed since the first successful human embryo conceived from a fertilization in vitro. Despite all advances, success rates in assisted reproduction techniques still remain unsatisfactory and it is well established that oxidative stress can be one of the major factors causing failure in in vitro fertilization (IVF) techniques. Recent Advances: In the past years, researchers have been shown details of the supportive role CCs play along oocyte maturation, development, and fertilization processes. Regarding redox metabolism, it is now evident that the synergism between gamete and somatic CCs is fundamental to further support a healthy embryo, since the oocyte lacks several defense mechanisms that are provided by the CCs. Critical Issues: There are many sources of reactive oxygen species (ROS) in the female reproductive tract in vivo that can be exacerbated (or aggravated) by pathological features. While an imbalance between ROS and antioxidants can result in oxidative damage, physiological levels of ROS are essential for oocyte maturation, ovulation, and early embryonic growth where they act as signaling molecules. At the event of an assisted reproduction procedure, the cumulus/oophorus complex is exposed to additional sources of oxidative stress in vitro. The cumulus cells (CCs) play essential roles in protecting the oocytes from oxidative damage. Future Directions: More studies are needed to elucidate redox biology in human CCs and oocyte. Also, randomized controlled trials will identify possible benefits of in vivo or in vitro administration of antioxidants for patients seeking IVF procedure. Antioxid. Redox Signal. 32, 522-535.

In vitro maturation (IVM) is an assisted reproduction technique with reduced hormone-related side effects. Several attempts to implement IVM in routine practice have failed, primarily due to its relatively low efficiency compared to conventional in vitro fertilization (IVF). Recently, capacitation (CAPA)-IVM, a novel two-step IVM method, has improved the embryology outcomes through synchronizing the oocyte nuclear and cytoplasmic maturation. However, the efficiency gap between CAPA-IVM and conventional IVF is still noticeable especially in the numerical production of good quality embryos. Considering the importance of glucose for oocyte competence, its metabolization is studied within both in vivo and CAPA-IVM matured mouse cumulus-oocyte-complexes (COCs) through direct measurements in both cellular compartments, from transcriptional and translational perspectives, to reveal metabolic shortcomings within the CAPA-IVM COCs. These results confirmed that within in vivo COC, cumulus cells are highly glycolytic, whereas oocytes, with low glycolytic activity, are deviating their glucose towards pentose phosphate pathway. No significant differences were observed in the CAPA-IVM oocytes compared to their in vivo counterparts. However, their cumulus cells exhibited a precocious increase of glycolytic activity during the pre-maturation culture step and activity was decreased during the IVM step. Here, specific alterations in mouse COC glucose metabolism due to CAPA-IVM culture were characterized using direct measurements for the first time. Present data show that, while CAPA-IVM cumulus cells are able to utilize glucose, their ability to support oocytes during final maturation is impaired. Future CAPA-IVM optimization strategies could focus on adjusting culture media energy substrate concentrations and/or implementing co-culture strategies.

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Establishing an ideal human follicle culture system for oncofertility patients relies mainly on animal models since donor tissue is scarce and often of suboptimal quality. The in vitro system developed in our laboratory supports the growth of prepubertal mouse secondary follicles up to mature oocytes. Given the importance of glucose in preparing the oocyte for proper maturation, a baseline characterization of follicle metabolism both in the culture system and in vivo was carried out. Markers of glucose-related pathways (glycolysis, tricarboxylic acid (TCA) cycle, pentose phosphate pathway (PPP), polyol pathway, hexosamine biosynthesis pathway (HBP)) as well as for the antioxidant capacity were measured in the different follicle cell types by both enzymatic activities (spectrophotometric detection) and gene expression (qPCR). This study confirmed that in vivo the somatic cells, mainly granulosa, exhibit intense glycolytic activity, while oocytes perform PPP. Throughout the final maturation step, oocytes in vivo and in vitro showed steady levels for all the key enzymes and metabolites. On the other hand, ovulation triggers a boost of pyruvate and lactate uptake in cumulus cells in vivo, consumes reduced nicotinamide adenine dinucleotide phosphate (NADPH) and increases TCA cycle and small molecules antioxidant capacity (SMAC) activities, while in vitro, the metabolic upregulation in all the studied pathways is limited. This altered metabolic pattern might be a consequence of cell exhaustion because of culture conditions, impeding cumulus cells to fulfil their role in providing proper support for acquiring oocyte competence. SUMMARY SENTENCE: In vitro cultured mouse follicles exhibit altered glycolytic activity and redox metabolism in the somatic compartment during meiotic maturation.

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Study question Is it possible to predict top quality embryos through gene expression analysis of cumulus cells and artificial intelligence before fertilization? Summary answer The artificial inteligence based tool OsteraTest is able to predict the ability of the oocyte to develop into a top quality blastocyst with 86% accuracy. What is known already Proper oocyte selection is an important bottleneck for In Vitro Fertilization (IVF) success. Nowadays, oocyte selection relies mainly in morphological analyses, which is not an unbiased method and may fail to reveal the real competence status of gametes. Cumulus oophorus cells (CC) are somatic cells that surround the oocyte at the antral follicle. It is directly involved in oocyte maturation and development, and thus is a valuable non-invasive source of biological information regarding the oocyte’s health. Artificial intelligence can be used to identify key biological processes and markers of interest through machine learning methods and could thus be applied. Study design, size, duration This is a prospective study that included data from 80 CC samples retrieved from publicly available microarray data (GSE27377) in the algorithm construction phase and 65 CC samples from each oocyte of 26 patients submitted to Intracytoplasmic Sperm Injection (ICSI) in validation phase. Samples were divided in two groups: CCs from oocytes that developed into top quality blastocysts in day 5 after ICSI and CCs from oocytes that presented arrested development. Participants/materials, setting, methods Samples were submitted to real time quantitative PCR with 25 target genes. Afterwards, gene expression levels for each gene and sample were submitted to the final algorithm, that was computed into a software, the OsteraTest, in a double-blind approach. The software indicated the development potential of each oocyte and this ranking was compared to the embryologist’s day 5 blastocyst classification according to Gardner. Main results and the role of chance The bioinformatic approach implemented resulted in the OsteraTest, composed of 8 machine learning models using a 25-gene network that altogether can predict oocyte quality, thus representing a very complex assembly. The software presented more than 86% accuracy in predicting the oocytes developmental capacity into a top-quality day 5 blastocyst. Top quality blastocysts present over 80% chance of resulting in a healthy pregnancy and live birth, and so this approach could be further used as a pregnancy potential predictor after a prospective study is conducted, analyzing CCs from oocytes that were further fertilized, developed into blastocysts and transferred in single embryo transfers. This tool can contribute greatly to improve success rates in IVF procedures and to assess egg quality in egg freezing procedures, providing information about the gametes potential even years before its use. Limitations, reasons for caution A large-scale, prospective, randomized study is necessary for further validation of these findings and to confirm the validity of the OsteraTest in the clinical environment. Such study is now being conducted in our lab. Wider implications of the findings The OsteraTest proved to be a valuable non-invasive tool to predict embryo formation and oocyte capacity even before fertilization.It can enable the clinics to anticipate successful treatments and provide a predictive report for oocyte freezing patients. Trial registration number #68081017.2.0000.5347

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.

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.